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Cockburn, I. A.

Publications and source records attributed to Cockburn, I. A..

18 recordsLinked to original sources
bioRxiv2026

Innate immune sensing of cell traversal by Plasmodium sporozoites drives protective T cell responses

Attenuated Plasmodium sporozoites elicit robust adaptive immune responses that protect against parasite challenge, yet how they engage the innate immune system remains poorly understood. To obtain sterile preparation of sporozoites we used flow cytometry to isolate pure GFP+ P. berghei and P. falciparum sporozoites, which were sorted onto murine bone marrow-derived macrophages (BMDMs) or human PBMCs to assess innate immune activation. Sporozoites induced a distinctive activation pattern resembling cell wounding responses, consistent with their ability to traverse host cells en route to the liver. Traversal-deficient sporozoites failed to activate BMDMs and showed impaired priming of protective CD8+ T cell immunity. This wounding response was independent of inflammasome or Toll-like receptor signaling but required {gamma}{delta} T cells, which are known to support CD8+ T cell responses against Plasmodium. These findings reveal a previously unappreciated innate sensing mechanism triggered by cell traversal that underpins the potent immunogenicity of Plasmodium sporozoites.

immunology↗
bioRxiv2025

Liver sinusoid constraints make random search of CD8 T cells for Plasmodium parasites efficient

A relatively small number of liver-localized CD8 T cells can provide sterilizing protection against exposure to Plasmodium sporozoites. Previously published mathematical model-based simulations of randomly searching for an infection T cells, assuming that T cells move in open (3D) space or on a regular lattice, suggested that some degree of attraction is needed for cells to find the infection. Yet, in our previous experiments we failed to find evidence of attraction towards the infection in most T cells. Because the movement of liver-localized T cells is confined to sinusoids, the blood vessels of the liver, T cells may in fact be attracted to the infection site via the sinusoids. In new experiments we imaged Plasmodium-specific CD8 T cells, sporozoites, and liver sinusoids simultaneously. We also developed a new method to model imaged sinusoids as graphs and to simulate movement of T cells on graphs, and new tests to detect biased cell movement on graphs. As before, our previously published open space/3D-based metric suggested that few T cells are attracted to the infection site in presence of moderate cluster of T cells. However, with our new graph-based metric we could not detect attraction when constraining T cell movement to sinusoids. Surprisingly, simulations of T cells searching for an infection via liver sinusoids showed that a relatively small number of T cells (1 cell/imaging volume or <10^5 cell/liver), moving without attraction at experimentally observed speeds, is sufficient to find the infection within 24 hours, well below the expected lifespan of the Plasmodium liver stage in mice. Our results suggest that the constrained environment of the tissue structure makes a random search more effective, and that detecting T cell attraction towards specific areas may arise as an artifact of metrics designed for open space/3D. Our results thus call for a re-evaluation of the implicit assumption that attraction is necessary for T cells to find infections in tissues.

immunology↗
bioRxiv2025

Conserved but mechanistically diverse piRNA defence against gypsy LTR retrotransposons in insect ovaries

Organisms have evolved highly adaptable defence systems to silence mobile genetic elements such as transposons. While individual transposons may emerge and vanish over time, the long-term evolutionary stability of the pathways that suppress them remains poorly understood. In the ovarian somatic cells of Drosophila, envelope-carrying gypsy long terminal repeat (LTR) retrotransposons are silenced by the Piwi-interacting RNA (piRNA) pathway, which depends exclusively on Piwi - an evolutionary derivative of an Aubergine/Piwi gene duplication unique to flies. Recent studies, however, have revealed that envelope-carrying gypsy elements are widespread across metazoans and particularly abundant in insect genomes, raising questions about the origin of ovarian somatic piRNA defence. Here we demonstrate that Aedes and Anopheles mosquitoes, as well as stingless bees and crickets, all produce piRNAs targeting gypsy elements in ovarian somatic cells, indicating a continuous arms race in this cellular niche for more than 400 million years of insect evolution. Notably, in Aedes aegypti, ovarian somatic cells express the same piRNA clusters as other somatic tissues, where they are known to target RNA viruses - suggesting a shared origin of anti-viral and anti-retrotransposon defences. Furthermore, we reveal striking lineage-specific differences in ovarian somatic piRNA biogenesis: slicing-independent phasing dominates in dipterans, ping-pong amplification in bees, and slicing-dependent phasing in crickets. Together, these findings indicate that distinct piRNA pathways have independently evolved at different timepoints to silence the same class of retrotransposons in insect evolution.

evolutionary biology↗
bioRxiv2024

Vgamma1+ gammadelta T cell-derived IL-4 initiates CD8 T cell immunity

Dendritic cells (DC) are pivotal for initiating adaptive immunity, a process triggered by the activation of DC via pathogen products or damage. Here, we describe an additional layer to this process, essential when pathogen-derived signals alone cannot directly achieve full DC activation. Immunisation with sporozoites from Plasmodium leads to CD8 T cell priming in a complex response that is initiated by a collaboration between conventional type 1 DC (cDC1) and {gamma}{delta} T cells. We unveil a pivotal initiating role for V{gamma}1+ {gamma}{delta} T cells, as they directly supply IL-4 to DC and CD8 T cells. IL-4 synergises with a CD4 T cell-derived CD40L signal to induce IL-12 production by cDC1. Both IL-12 and IL-4 then directly signal CD8 T cells, with synergy between these cytokines driving enhanced IL-12 receptor expression and expansion of responding CD8 T cells. This study reveals a key role for V{gamma}l+ {gamma}{delta} T cells in initiating CD8 T cell immunity to Plasmodium. More broadly, it shows that responses to some pathogens require help from innate-like T cells to pass an initiation threshold and further amplify the response in a process underscored by IL-4 production.

immunology↗
bioRxiv2024

B cells targeting parasites capture spatially linked antigens to secure T cell help

Our understanding of T-cell-dependent humoral responses has been largely shaped by studies involving model antigens such as recombinant proteins and viruses 1,2. In these contexts, B cells internalize the entire antigen or pathogen, and present a range of antigens to helper CD4+ T cells to initiate the humoral response. However, this model does not account for large pathogens (such as parasites) that are too large to be taken up by individual B cells, and the mechanisms by which B cells acquire and present antigens from large complex pathogens to T cells remain poorly understood. Here we used Plasmodium, the causative parasite of malaria, as a model to investigate the requirements for T cell help for B cells targeting the Plasmodium surface circumsporozoite protein (CSP). Upon Plasmodium sporozoite (SPZ) immunization, CSP-specific B cells can form a synapse-like structure with SPZs and take up CSP and non-CSP surface antigens. As a result, CSP-specific B cells can receive help from CD4+ T cells specific to antigens that are located on the surface but not cytosol of the Plasmodium SPZ. Therefore, B cells can obtain help, not only from T cells with the same protein specificity, but also from T cells specific for spatially linked antigens. This flexibility in T cell help may enhance the initiation and maintenance of humoral immune responses to complex pathogens.

immunology↗
bioRxiv2023

Plasma cells are formed in waning germinal centers via an affinity-independent process

Long-lived plasma cells (PCs) secrete antibodies that can provide sustained immunity against infection. It has been proposed that high affinity cells are preferentially selected into this compartment, potentiating the immune response. Here we used single cell RNA-seq to track the development of Ighg2A10 cells, specific for the Plasmodium falciparum circumsporozoite protein (PfCSP) within the germinal center (GC). We identified cells differentiating into memory B cells (MBC) or PCs and estimated their affinity by V(D)J sequencing. While pre-memory cells were of lower affinity than GC B cells generally, the affinity of pre-PC cells was indistinguishable. Rather, a larger proportion of cells differentiated into PCs in waning GCs. These later emigrants replaced early arrivals in the bone marrow allowing the development of a high affinity PC compartment.

immunology↗
bioRxiv2022

ZEB2 regulates the development of CD11c+ atypical B cells

CD11c+ atypical B cells (ABC) are an alternative memory B cell lineage identified both in normal immune responses as well as pathogenic responses in autoimmunity. While it is clear that ABCs have a distinct transcriptional program, the factors that direct this program have not been identified. Here, we generated a human tonsil single-cell RNA-seq dataset and identified candidate transcription factors associated with the ABC population. We selected 8 of these transcription factors for further analysis based on their conserved expression in mouse ABC bulk RNA-seq datasets. Using an optimized CRSPR-Cas9 knockdown method we found that only zinc finger E-box binding homeobox 2 (Zeb2) knock-out impaired ABC formation. To assess the role of Zeb2 in ABC formation in vivo we used Zeb2fl/fl mice crossed to a CD23Cre line. Germinal center and plasma cell responses in these mice after Plasmodium sporozoite immunization were largely unaltered but we observed a specific defect in ABC formation. We further determined that ZEB2 haploinsufficient Mowat Wilson syndrome patients also have decreased circulating ABCs in the blood, supporting a role for this transcription factor in humans as well as mice. In sum, we identified Zeb2 as a key TF governing the formation of ABCs.

immunology↗
bioRxiv2022

Mathematical modeling suggests that a single Plasmodium-specific CD8 T cell can eliminate a malaria liver stage in mice

Vaccination strategies in mice inducing high numbers of memory CD8+ T cells specific to a single epitope are able to provide sterilizing protection against infection with Plasmodium sporozoites. We have recently found that Plasmodium-specific CD8+ T cells cluster around sporozoite-infected hepatocytes but whether such clusters are important in elimination of the parasite remains incompletely understood. Here we used our previously generated data in which we employed intravital microscopy to longitudinally image 32 GFP-expressing Plasmodium yoelii parasites in livers of mice that had received activated Plasmodium-specific CD8+ T cells after sporozoite infection. We found significant heterogeneity in the dynamics of the normalized GFP signal from the parasites (termed "vitality index" or VI) that was weakly correlated with the number of T cells near the parasite. We also found that a simple model assuming mass-action, additive killing by T cells well describes the VI dynamics for most parasites and predicts a highly variable killing efficacy by individual T cells. Given our estimated median per capita kill rate of k = 0.031/h we predict that a single T cell is typically incapable to kill a parasite within the 48 hour lifespan of the liver stage in mice. Stochastic simulations of T cell clustering and killing of the liver stage also suggested that 1) three or more T cells per infected hepatocyte are required to ensure sterilizing protection; 2) both variability in killing efficacy of individual T cells and resistance to killing by individual parasites may contribute to the observed variability in VI decline, and 3) the stable VI of some clustered parasites cannot be explained by measurement noise. Taken together, our analysis for the first time provides estimates of efficiency at which individual CD8+ T cells eliminate intracellular parasitic infection in vivo.

immunology↗
bioRxiv2021

Platelets are dispensable for the ability of CD8+ T cells to accumulate, patrol, kill and reside in the liver

Effector and memory CD8+ T cells accumulate in large numbers in the liver where they play key roles in the control of liver pathogens including Plasmodium. It has also been proposed that liver may act as the main place for elimination of effector CD8+ T cells at the resolution of immune responses. Platelets and the integrin LFA-1 have been proposed to be critical for the accumulation of protective CD8+ T cells in the liver; conversely, asialo-glycoprotein (ASGP) expression on the surface of CD8+ T cells has been proposed to assist in elimination of effector T cells in the liver. Here we investigated the contributions of these interactions in the accumulation of CD8+ T cells activated in vitro or in vivo by immunization with Plasmodium parasites. Using Mpl-/- mice with constitutive thrombocytopaenia and antibody-mediated platelet depletion models we found that severe reduction in platelet concentration in circulation did not strongly influence the accumulation and protective function of CD8+ T cells in the liver in these models. Surprisingly, inhibition of ASGP receptors did not inhibit the accumulation of effector cells in the liver, but instead prevented these cells from accumulating in the spleen. We further found that enforced expression of ASGP on effector CD8+ T cells using ST3GalI knockout cells lead to their loss from the spleen. These data suggest that platelets play a marginal role in CD8+ T cell function in the liver. Furthermore, ASGP-expressing effector CD8+ T cells are retained in the liver but are lost from the spleen.

immunology↗
bioRxiv2021

Dysregulation of PAX5 causes uncommitted B cell development and tumorigenesis in mice

PAX5 is the master transcription factor controlling B cell identity. In humans, mutations in PAX5 account for 30% of B cell acute lymphoblastic leukemia (B-ALL) cases. Investigating the causal effects of PAX5 mutations has however been difficult due to the premature lethality of Pax5-/- mice. Here we describe a novel mouse strain with a premature STOP mutation in Pax5 (Y351*) that produces a truncated protein and reduction in protein function, yet still allows for some B cell development to occur. A population of uncommitted and multipotent CD19+B220- B cells develops in the bone marrow of homozygous mice leading to the development of B-ALL. We show that the tumors frequently acquire secondary mutations in Jak3, and Ptpn11 highlighting key pathways interacting with PAX5 during malignant transformation. Analysis of the PAX5Y351* mice provide insight not only into the functional consequence of reduced PAX5 activity on B cell development and identity, but also provides an avenue in which to study PAX5-driven B-ALL in mice. One Sentence SummaryReduction in PAX5 function in mice induces the development of uncommitted B cells that have multipotent and malignant potential.

immunology↗
bioRxiv2020

A minority of liver-localized CD8 T cells demonstrate difficult-to-detect attraction to Plasmodium-infected hepatocytes

Malaria is a disease caused by Plasmodium parasites, resulting in over 200 million infections and 400,000 deaths every year. A critical step of malaria infection is when sporozoites, injected by mosquitoes, travel to the liver and form liver stages. Malaria vaccine candidates which induce large numbers of malaria-specific CD8 T cells in mice are able to eliminate all liver stages, preventing fulminant malaria. However, how CD8 T cells find all parasites in 48 hours of the liver stage lifespan is not well understood. Using intravital microscopy of murine livers, we generated unique data on T cell search for malaria liver stages within a few hours after infection. To detect attraction of T cells to an infection site, we used the von Mises-Fisher distribution in 3D, similar to the 2D von Mises distribution previously used in ecology. Our results suggest that the vast majority (70-95%) of malaria-specific and non-specific liver-localized CD8 T cells did not display attraction towards the infection site, suggesting that the search for malaria liver stages occurs randomly. However, a small fraction (15-20%) displayed weak but detectable attraction towards parasites which already had been surrounded by several T cells. We found that speeds and turning angles correlated with attraction, suggesting that understanding mechanisms that determine the speed of T cell movement in the liver may improve the efficacy of future T cell-based vaccines. Stochastic simulations suggest that a small movement bias towards the parasite dramatically reduces the number of CD8 T cells needed to eliminate all malaria liver stages, but to detect such attraction by individual cells requires data from long imaging experiments which are not currently feasible. Importantly, as far as we know this is the first demonstration of how activated/memory CD8 T cells might search for the pathogen in nonlymphoid tissues a few hours after infection. We have also established a framework for how attraction of individual T cells towards a location in 3D can be rigorously evaluated.

immunology↗
bioRxiv2020

Environment-imposed constraints make Brownian walkers efficient searchers

Pathogen-specific CD8 T cells face the problem of finding rare cells that present their cognate antigen either in the lymph node or infected tissue. While quantitative details of T cell movement strategies in some tissues such lymph nodes or skin have been relatively well characterized, we still lack quantitative understanding of T cell movement in many other important tissues such as the spleen, lung, liver, and gut. Furthermore, how environmental constraints influence movement patterns of T cells in tissues remains incompletely characterized. Liver is one of the major organs in mice and humans and is the target site for many pathogens such as hepatitis B and C viruses in humans and Plasmodium parasites in multiple mammalian species. We developed a protocol to generate stable numbers of liver-located CD8 T cells, used intravital microscopy to record movement patterns of CD8 T cells in livers of live mice, and analyzed these and previously published data using well-established statistical and computational methods. We show that in most of our experiments Plasmodium-specific liver-localized CD8 T cells perform correlated random walks characterized by transiently superdiffusive displacement with persistence times of 10-15 min that exceed those observed for T cells in lymph nodes. Liver-localized CD8 T cells typically crawl on the lumenal side of liver sinusoids (i.e., are in the blood); simulating T cell movement in digital structures derived from the liver sinusoids illustrates that liver structure alone is sufficient to explain the relatively long superdiffusive displacement of T cells. In experiments when CD8 T cells in the liver poorly attach to the sinusoids (e.g., one week post immunization with radiation-attenuated Plasmodium sporozoites) T cells also undergo Levy flights - large displacements occurring due to cell deattaching from the endothelium, floating with the blood flow, and re-attaching at another location. Our analysis, thus, provides quantitative details of movement patterns of liver-localized CD8 T cells and illustrates how structural and physiological details of the tissue may impact T cell movement patterns.

immunology↗
bioRxiv2020

PD-L1hi plasmablasts limit the T cell response during the acute phase of parasite infections

During infections with protozoan parasites or virus, T cell immunosuppression is generated simultaneously with a high B cell activation. Here, we show that in T. cruzi infection, all plasmablasts detected had higher surface expression of PD-L1, than other mononuclear cells. PD-L1hi plasmablasts were induced in vivo in an antigen-specific manner and required help from Bcl-6+CD4+T cells. PD-L1hi expression was not a characteristic of all antibody-secreting cells since plasma cells found during the chronic phase of infection express PD-L1 but at lower levels. PD-L1hi plasmablasts were also present in mice infected with Plasmodium or with lymphocytic choriomeningitis virus, but not in mice with autoimmune disorders or immunized with T cell-dependent antigens. PD-L1hi plasmablasts suppressed T cell response, via PD-L1, in vitro and in vivo. Thus, this study reveals that extrafollicular PD-L1hi plasmablasts, which precede the germinal center (CG) response, are a suppressive population in infections that may influence T cell response. Brief summaryPathogens develop different strategies to settle in the host. We identified a plasmablats population induced by pathogens in acute infections which suppress T cell response.

immunology↗
bioRxiv2020

Atypical B cells are a normal component of immune responses to vaccination and infection in humans

The full diversity of the circulating human B cell compartment is unknown. Flow cytometry analysis suggests that in addition to naive and memory B cells, there exists a population of CD11c+, CD27- CD21- "atypical" B cells, that are associated with chronic or recurrent infection and autoimmunity. We used single cell RNA-seq approaches to examine the diversity of both antigen-specific B cells and total B cells in healthy subjects and individuals naturally-exposed to recurrent malaria infections. This analysis revealed two B cell lineages: a classical lineage of activated and resting memory B cells, and an atypical-like lineage. Surprisingly, the atypical lineage was common in both malaria exposed individuals and non-exposed healthy controls. Using barcoded antibodies in conjunction with our transcriptomic data, we found that atypical lineage cells in healthy individuals lack many atypical B markers and thus represent an undercounted cryptic population. We further determined using antigen specific probes that atypical cells can be induced by primary vaccination in humans and can be recalled upon boosting. Collectively these data suggest that atypical cells are not necessarily pathogenic but can be a normal component of B responses to antigen.

immunology↗
bioRxiv2020

Avid binding by B cells to the Plasmodium circumsporozoite protein repeat suppresses responses to protective subdominant epitopes

Antibodies targeting the NANP/NVDP repeat domain of the Plasmodium falciparum circumsporozoite protein (CSPRepeat) can confer protection against malaria. However, it has also been suggested that this repeat domain exists as a decoy that distracts the immune system from mounting protective responses targeting other domains of CSP. Here we show that B cell responses to the repeat domain are indeed [~]10 fold higher than responses to the N- and C-terminal regions of CSP after sporozoite immunization. We investigated the role of the number of CSPRepeat-specific naive precursor B cells and high avidity binding by B cells in driving the immunodominance of the CSPRepeat. Using adoptive transfer of germline precursors specific for the CSPRepeat, we found that increasing precursor number did indeed increase the responses to the repeat region, but not to the detriment of responses to other epitopes. To investigate the role of avid binding by B cells to the CSPRepeat in driving immunodominance we generated CSP9: a truncated CSP molecule with just 9 NANP repeats. Compared to near full length CSP molecules, CSP9 induced lower BCR signalling in CSPRepeat-specific cells and induced stronger responses to non-repeat epitopes. Finally, we found mice immunized with CSP9 molecules were strongly protected against mosquito bite challenge. Collectively these data demonstrate that the CSPRepeat does function as an immunodominant decoy and that truncated CSP molecules may be a promising avenue for future malaria vaccines. Significance StatementMalaria kills approximately 420,000 individuals each year(1). Our best vaccine, RTS,S/AS01 is based on the circumsporozoite protein that coasts the surface of the parasite. However, this vaccine is only partially protective. Here we show that responses to a repeat region in the circumsporozoite dominate the immune response. However, immunizing with a circumsporozoite protein with a shortened repeat region induces a more diverse immune response, which could be an avenue to make better malaria vaccines.

immunology↗
bioRxiv2019

Antibody feedback limits the expansion of cognate memory B cells but drives the diversification of vaccine-induced antibody responses

Generating sufficient antibody to block infection is a key challenge for vaccines against malaria. Here we show that antibody titres to a key target, the repeat region of the Plasmodium falciparum circumsporozoite protein (PfCSP), plateaued after two immunizations in a clinical trial of the radiation-attenuated sporozoite vaccine. To understand the mechanisms limiting vaccine responsiveness, we developed Ig-knockin mice with elevated numbers of PfCSP-binding B cells. We determined that recall responses were inhibited by antibody feedback via epitope masking of the immunodominant PfCSP repeat region. Importantly, the amount of antibody that prevents boosting is below the amount of antibody required for protection. Finally, while antibody feedback limited responses to the PfCSP-repeat region in vaccinated volunteers, potentially protective subdominant responses to C-terminal regions did expand with subsequent boosts. These data suggest that antibody feedback drives the diversification of immune responses and that vaccination for malaria will require the targeting of multiple antigens.

immunology↗
bioRxiv2017

Multivalency drives the neutralizing activity of antibodies against the Plasmodium falciparum circumsporozoite protein

The repeat region of the Plasmodium falciparum circumsporozoite protein (CSP) is a major vaccine antigen because it can be targeted by parasite neutralizing antibodies; however, little is known about this interaction. We used isothermal titration calorimetry, X-ray crystallography and mutagenesis-validated modeling to analyze the binding of a murine neutralizing antibody to Plasmodium falciparum CSP. Strikingly, we found that the repeat region of CSP is bound by multiple antibodies. This repeating pattern allows multiple weak interactions of single FAB domains to accumulate and yield a complex with a dissociation constant in the low nM range. Because the CSP protein can potentially cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T cell independent. However, while there was a modest response in mice deficient in T cell help, the bulk of the response was T cell dependent. By sequencing the BCRs of CSP-repeat specific B cells in inbred mice we found that these cells underwent somatic hypermutation and affinity maturation indicative of a T-dependent response. Last, we found that the BCR repertoire of responding B cells was limited suggesting that the structural simplicity of the repeat may limit the breadth of the immune response.\n\nAuthor SummaryVaccines aim to protect by inducing the immune system to make molecules called antibodies that can recognize molecules on the surface of invading pathogens. In the case of malaria, our most advanced vaccine candidates aim to promote the production of antibodies that recognize the circumsporozoite protein (CSP) molecule on the surface of the invasive parasite stage called the sporozoite. In this report we use X-ray crystallography to determine the structure of CSP-binding antibodies at the atomic level. We use other techniques such as isothermal titration calorimetry and structural modeling to examine how this antibody interacts with the CSP molecule. Strikingly, we found that each CSP molecule could bind 6 antibodies. This finding has implications for the immune response and may explain why high titers of antibody are needed for protection. Moreover, because the structure of the CSP repeat is quite simple we determined that the number of different kinds of antibodies that could bind this molecule are quite small. However a high avidity interaction between those antibodies and CSP can result from a process called affinity maturation that allows the body to learn how to make improved antibodies specific for pathogen molecules. These data show that while it is challenging for the immune system to recognize and neutralize CSP, it should be possible to generate viable vaccines targeting this molecule.

immunology↗