Migration-induced cell shattering due to DOCK8 deficiency causes a type 2-biased helper T cell response.

Mutations that impact immune cell migration and result in immune deficiency illustrate the importance of cell movement in host defense. In humans, loss-of-function mutations in DOCK8, a guanine exchange factor involved in hematopoietic cell migration, lead to immunodeficiency and, paradoxically, allergic disease. Here, we demonstrate that, like humans, Dock8-/- mice have a profound type 2 CD4+ helper T (TH2) cell bias upon pulmonary infection with Cryptococcus neoformans and other non-TH2 stimuli. We found that recruited Dock8-/-CX3CR1+ mononuclear phagocytes are exquisitely sensitive to migration-induced cell shattering, releasing interleukin (IL)-1ß that drives granulocyte-macrophage colony-stimulating factor (GM-CSF) production by CD4+ T cells. Blocking IL-1ß, GM-CSF or caspase activation eliminated the type-2 skew in mice lacking Dock8. Notably, treatment of infected wild-type mice with apoptotic cells significantly increased GM-CSF production and TH2 cell differentiation. This reveals an important role for cell death in driving type 2 signals during infection, which may have implications for understanding the etiology of type 2 CD4+ T cell responses in allergic disease.

Reservoir host immune responses to emerging zoonotic viruses.

Zoonotic viruses, such as HIV, Ebola virus, coronaviruses, influenza A viruses, hantaviruses, or henipaviruses, can result in profound pathology in humans. In contrast, populations of the reservoir hosts of zoonotic pathogens often appear to tolerate these infections with little evidence of disease. Why are viruses more dangerous in one species than another? Immunological studies investigating quantitative and qualitative differences in the host-virus equilibrium in animal reservoirs will be key to answering this question, informing new approaches for treating and preventing zoonotic diseases. Integrating an understanding of host immune responses with epidemiological, ecological, and evolutionary insights into viral emergence will shed light on mechanisms that minimize fitness costs associated with viral infection, facilitate transmission to other hosts, and underlie the association of specific reservoir hosts with multiple emerging viruses. Reservoir host studies provide a rich opportunity for elucidating fundamental immunological processes and their underlying genetic basis, in the context of distinct physiological and metabolic constraints that contribute to host resistance and disease tolerance.

Focusing in on T cell cross-reactivity.

To provide broad immunity to a vast array of foreign antigens with a limited number of T lymphocytes, each cell has to recognize many targets. By implementing a strategy to identify T cell receptor (TCR) ligands and investigating at a fine granularity their structure and sequence relationship, Birnbaum et al. demonstrate the surprisingly tight focus of such T cell cross-reactivity.

Chronic infection control relies on T cells with lower foreign antigen binding strength generated by N-nucleotide diversity.

The breadth of pathogens to which T cells can respond is determined by the T cell receptors (TCRs) present in an individual's repertoire. Although more than 90% of the sequence diversity among TCRs is generated by terminal deoxynucleotidyl transferase (TdT)-mediated N-nucleotide addition during V(D)J recombination, the benefit of TdT-altered TCRs remains unclear. Here, we computationally and experimentally investigated whether TCRs with higher N-nucleotide diversity via TdT make distinct contributions to acute or chronic pathogen control specifically through the inclusion of TCRs with lower antigen binding strengths (i.e., lower reactivity to peptide-major histocompatibility complex (pMHC)). When T cells with high pMHC reactivity have a greater propensity to become functionally exhausted than those of low pMHC reactivity, our computational model predicts a shift toward T cells with low pMHC reactivity over time during chronic, but not acute, infections. This TCR-affinity shift is critical, as the elimination of T cells with lower pMHC reactivity in silico substantially increased the time to clear a chronic infection, while acute infection control remained largely unchanged. Corroborating an affinity-centric benefit for TCR diversification via TdT, we found evidence that TdT-deficient TCR repertoires possess fewer T cells with weaker pMHC binding strengths in vivo and showed that TdT-deficient mice infected with a chronic, but not an acute, viral pathogen led to protracted viral clearance. In contrast, in the case of a chronic fungal pathogen where T cells fail to clear the infection, both our computational model and experimental data showed that TdT-diversified TCR repertoires conferred no additional protection to the hosts. Taken together, our in silico and in vivo data suggest that TdT-mediated TCR diversity is of particular benefit for the eventual resolution of prolonged pathogen replication through the inclusion of TCRs with lower foreign antigen binding strengths.

What's self got to do with it: Sources of heterogeneity among naive T cells.

There is a long-standing assumption that naive CD4+ and CD8+ T cells are largely homogeneous populations despite the extraordinary diversity of their T cell receptors (TCR). The self-immunopeptidome plays a key role in the selection of the naive T cell repertoire in the thymus, and self-peptides are also an important driver of differences between individual naive T cells with regard to their subsequent functional contributions to an immune response. Accumulating evidence suggests that as early as the ß-selection stage of T cell development, when only one of the recombined chains of the mature TCR is expressed, signaling thresholds may be established for positive selection of immature thymocytes. Stochastic encounters subsequently made with self-ligands during positive selection in the thymus imprint functional biases that a T cell will carry with it throughout its lifetime, although ongoing interactions with self in the periphery ensure a level of plasticity in the gene expression wiring of naive T cells. Identifying the sources of heterogeneity in the naive T cell population and which functional attributes of T cells can be modulated through post-thymic interventions versus those that are fixed during T cell development, could enable us to better select or generate T cells with particular traits to improve the efficacy of T cell therapies.

Nuclear segmentation facilitates neutrophil migration.

Neutrophils are among the fastest-moving immune cells. Their speed is critical to their function as 'first responder' cells at sites of damage or infection, and it has been postulated that the unique segmented nucleus of neutrophils functions to assist their rapid migration. Here, we tested this hypothesis by imaging primary human neutrophils traversing narrow channels in custom-designed microfluidic devices. Individuals were given an intravenous low dose of endotoxin to elicit recruitment of neutrophils into the blood with a high diversity of nuclear phenotypes, ranging from hypo- to hyper-segmented. Both by cell sorting of neutrophils from the blood using markers that correlate with lobularity and by directly quantifying the migration of neutrophils with distinct lobe numbers, we found that neutrophils with one or two nuclear lobes were significantly slower to traverse narrower channels, compared to neutrophils with more than two nuclear lobes. Thus, our data show that nuclear segmentation in primary human neutrophils provides a speed advantage during migration through confined spaces.

Revisiting thymic positive selection and the mature T cell repertoire for antigen.

To support effective host defense, the T cell repertoire must balance breadth of recognition with sensitivity for antigen. The concept that T lymphocytes are positively selected in the thymus is well established, but how this selection achieves such a repertoire has not been resolved. Here we suggest that it is direct linkage between self and foreign antigen recognition that produces the necessary blend of TCR diversity and specificity in the mature peripheral repertoire, enabling responses to a broad universe of unpredictable antigens while maintaining an adequate number of highly sensitive T cells in a population of limited size. Our analysis also helps to explain how diversity and frequency of antigen-reactive cells in a T cell repertoire are adjusted in animals of vastly different size scale to enable effective antipathogen responses and suggests a possible binary architecture in the TCR repertoire that is divided between germline-related optimal binding and diverse recognition.

CD5 levels define functionally heterogeneous populations of naïve human CD4+ T cells.

Studies in murine models show that subthreshold TCR interactions with self-peptide are required for thymic development and peripheral survival of naïve T cells. Recently, differences in the strength of tonic TCR interactions with self-peptide, as read-out by cell surface levels of CD5, were associated with distinct effector potentials among sorted populations of T cells in mice. However, whether CD5 can also be used to parse functional heterogeneity among human T cells is less clear. Our study demonstrates that CD5 levels correlate with TCR signal strength in human naïve CD4+ T cells. Further, we describe a relationship between CD5 levels on naïve human CD4+ T cells and binding affinity to foreign peptide, in addition to a predominance of CD5hi T cells in the memory compartment. Differences in gene expression and biases in cytokine production potential between CD5lo and CD5hi naïve human CD4+ T cells are consistent with observations in mice. Together, these data validate the use of CD5 surface levels as a marker of heterogeneity among human naïve CD4+ T cells with important implications for the identification of functionally biased T- cell populations that can be exploited to improve the efficacy of adoptive cell therapies.

T cell-positive selection uses self-ligand binding strength to optimize repertoire recognition of foreign antigens.

Developing T cells express diverse antigen receptors whose specificities are not prematched to the foreign antigens they eventually encounter. Past experiments have revealed that thymocytes must productively signal in response to self antigens to mature and enter the peripheral T cell pool (positive selection), but how this process enhances effective mature T cell responses to foreign antigen is not fully understood. Here we have documented an unsuspected connection between thymic recognition events and foreign antigen-driven T cell responses. We find that the strength of self-reactivity is a clone-specific property unexpectedly directly related to the strength of T cell receptor (TCR) binding to presented foreign antigen. T cells with receptors showing stronger interaction with self dominate in responses to infections and accumulate in aging individuals, revealing that positive selection contributes to effective immunity by skewing the mature TCR repertoire toward highly effective recognition of pathogens that pose a danger to the host.

Differential interferon-gamma production potential among naïve CD4+ T cells exists prior to antigen encounter.

Individual CD4+ T cells can become one of a number of helper (Th) lineages with distinct effector functions. However, whether biases in Th potential exist prior to antigen encounter is unknown. Studies have identified cell-intrinsic functional heterogeneity among naïve T cells that can be parsed based on the strength of T-cell receptor (TCR) interactions with self-peptide. Here, using CD5 levels as a surrogate for the strength of these basal TCR signals, we sought to identify pre-existing effector biases in the CD4+ T-cell lineage. We show that ex vivo-activated CD5lo CD4+ T cells produce greater amounts of the Th1 cytokine interferon-gamma (IFNγ) than their CD5hi counterparts. In addition, a greater percentage of CD5lo effector CD4+ T cells produce IFNγ in both polyclonal and monoclonal CD4+ T-cell populations after antigen challenge in vivo. These results suggest that differential IFNγ production potential exists among CD4+ T cells prior to activation and independent of TCR affinity for foreign antigen.

Regulatory T cells differ from conventional CD4+ T cells in their recirculatory behavior and lymph node transit times.

Regulatory T cells (Tregs) continuously suppress autoreactive immune responses within tissues to prevent autoimmunity, yet the recirculatory behavior of Tregs between and within tissues enabling the maintenance of peripheral tolerance remains incompletely defined. Here, we quantified homing efficiency to and the dwell time of Tregs within secondary lymphoid organs (SLOs) and used intravital two-photon microscopy to measure Treg surveillance behavior of dendritic cells. Tregs homed substantially less efficiently to SLOs compared with conventional CD4+ T cells (Tconvs), despite similar expression of homing receptors. Tregs remained on average 2-3 times longer within the LN than Tconvs before exiting, and retained Tregs differed from recirculating Tregs in phenotype, motility and interaction duration with dendritic cells. Taken together, these data revealed fundamental differences in Treg versus conventional T cell in vivo recirculation and migration behaviors, identified a Treg population with prolonged LN dwell time, and provided quantitative insight into their spatiotemporal behavior within LNs.

The Reticular Cell Network: A Robust Backbone for Immune Responses.

Lymph nodes are meeting points for circulating immune cells. A network of reticular cells that ensheathe a mesh of collagen fibers crisscrosses the tissue in each lymph node. This reticular cell network distributes key molecules and provides a structure for immune cells to move around on. During infections, the network can suffer damage. A new study has now investigated the network's structure in detail, using methods from graph theory. The study showed that the network is remarkably robust to damage: it can still support immune responses even when half of the reticular cells are destroyed. This is a further important example of how network connectivity achieves tolerance to failure, a property shared with other important biological and nonbiological networks.

Alterations in the Thymic Selection Threshold Skew the Self-Reactivity of the TCR Repertoire in Neonates.

Neonatal and adult T cells differ in their effector functions. Although it is known that cell-intrinsic differences in mature T cells contribute to this phenomenon, the factors involved remain unclear. Given emerging evidence that the binding strength of a TCR for self-peptide presented by MHC (self-pMHC) impacts T cell function, we sought to determine whether altered thymic selection influences the self-reactivity of the TCR repertoire during ontogeny. We found that conventional and regulatory T cell subsets in the thymus of neonates and young mice expressed higher levels of cell surface CD5, a surrogate marker for TCR avidity for self-pMHC, as compared with their adult counterparts, and this difference in self-reactivity was independent of the germline bias of the neonatal TCR repertoire. The increased binding strength of the TCR repertoire for self-pMHC in neonates was not solely due to reported defects in clonal deletion. Rather, our data suggest that thymic selection is altered in young mice such that thymocytes bearing TCRs with low affinity for self-peptide are not efficiently selected into the neonatal repertoire, and stronger TCR signals accompany both conventional and regulatory T cell selection. Importantly, the distinct levels of T cell self-reactivity reflect physiologically relevant differences based on the preferential expansion of T cells from young mice to fill a lymphopenic environment. Therefore, differences in thymic selection in young versus adult mice skew the TCR repertoire, and the relatively higher self-reactivity of the T cell pool may contribute to the distinct immune responses observed in neonates.

Weakly self-reactive T-cell clones can homeostatically expand when present at low numbers.

T-cell division is central to maintaining a stable T-cell pool in adults. It also enables T-cell expansion in neonates, and after depletion by chemotherapy, bone marrow transplantation, or infection. The same signals required for T-cell survival in lymphoreplete settings, IL-7 and T-cell receptor (TCR) interactions with self-peptide MHC (pMHC), induce division when T-cell numbers are low. The strength of reactivity for self-pMHC has been shown to correlate with the capacity of T cells to undergo lymphopenia-induced proliferation (LIP), in that weakly self-reactive T cells are unable to divide, implying that T-cell reconstitution would significantly skew the TCR repertoire toward TCRs with greater self-reactivity and thus compromise T-cell diversity. Here, we show that while CD4+ T cells with low self-pMHC reactivity experience more intense competition, they are able to divide when present at low enough cell numbers. Thus, at physiological precursor frequencies CD4+ T cells with low self-pMHC reactivity are able to contribute to the reconstitution of the T-cell pool.

IDO-orchestrated crosstalk between pDCs and Tregs inhibits autoimmunity.

Plasmacytoid dendritic cells (pDCs) have been shown to both mediate and prevent autoimmunity, and the regulation of their immunogenic versus tolerogenic functions remains incompletely understood. Here we demonstrate that, compared to other cells, pDCs are the major expressors of Indoleamine-2,3-dioxygenase (IDO) in steady-state lymph nodes (LNs). IDO expression by LN pDCs was closely dependent on MHCII-mediated, antigen-dependent, interactions with Treg. We further established that IDO production by pDCs was necessary to confer suppressive function to Tregs. During EAE development, IDO expression by pDCs was required for the generation of Tregs capable of dampening the priming of encephalitogenic T cell and disease severity. Thus, we describe a novel crosstalk between pDCs and Tregs: Tregs shape tolerogenic functions of pDCs prior to inflammation, such that pDCs in turn, promote Treg suppressive functions during autoimmunity.

Random migration and signal integration promote rapid and robust T cell recruitment.

To fight infections, rare T cells must quickly home to appropriate lymph nodes (LNs), and reliably localize the antigen (Ag) within them. The first challenge calls for rapid trafficking between LNs, whereas the second may require extensive search within each LN. Here we combine simulations and experimental data to investigate which features of random T cell migration within and between LNs allow meeting these two conflicting demands. Our model indicates that integrating signals from multiple random encounters with Ag-presenting cells permits reliable detection of even low-dose Ag, and predicts a kinetic feature of cognate T cell arrest in LNs that we confirm using intravital two-photon data. Furthermore, we obtain the most reliable retention if T cells transit through LNs stochastically, which may explain the long and widely distributed LN dwell times observed in vivo. Finally, we demonstrate that random migration, both between and within LNs, allows recruiting the majority of cognate precursors within a few days for various realistic infection scenarios. Thus, the combination of two-scale stochastic migration and signal integration is an efficient and robust strategy for T cell immune surveillance.

Quantification of lymph node transit times reveals differences in antigen surveillance strategies of naive CD4+ and CD8+ T cells.

Naïve T cells continually recirculate between blood and secondary lymphoid organs, scanning dendritic cells (DC) for foreign antigen. Despite its importance for understanding how adaptive immune responses are efficiently initiated from rare precursors, a detailed quantitative analysis of this fundamental process has not been reported. Here we measure lymph node (LN) entry, transit, and exit rates for naïve CD4(+) and CD8(+) T cells, then use intravital imaging and mathematical modeling to relate cell-cell interaction dynamics to population behavior. Our studies reveal marked differences between CD4(+) vs. CD8(+) T cells. CD4(+) T cells recirculate more rapidly, homing to LNs more efficiently, traversing LNs twice as quickly, and spending ∼1/3 of their transit time interacting with MHCII on DC. In contrast, adoptively transferred CD8(+) T cells enter and leave the LN more slowly, with a transit time unaffected by the absence of MHCI molecules on host cells. Together, these data reveal an unexpectedly asymmetric role for MHC interactions in controlling CD4(+) vs. CD8(+) T lymphocyte recirculation, as well as distinct contributions of T cell receptor (TCR)-independent factors to the LN transit time, exposing the divergent surveillance strategies used by the two lymphocyte populations in scanning for foreign antigen.

The race for the prize: T-cell trafficking strategies for optimal surveillance.

The initiation of T-cell responses requires rare precursors to locate a draining lymph node (dLN) and encounter dendritic cells (DCs) presenting peptide-major histocompatibility complexes (pMHCs). To locate this needle in the haystack rapidly, T cells face an optimization problem-what is the most efficient trafficking strategy for surveillance and recirculation through blood? Two extremes are scanning low numbers of DCs per node with frequent recirculation, or meticulous surveillance with infrequent recirculation. Naive T cells also require stimulation by self-pMHCs. To enable efficient location of both foreign and self, has evolution settled on an optimum time for T cells to spend surveying each lymph node? Using a data-driven mathematical model, we show the most efficient strategy for detecting antigen in a dLN depends on its abundance. Detection of low-density antigen is optimized with systemically slow transit. In contrast, at high densities or if dLN egress is restricted, rapid transit through other nodes is optimal. We argue that blood-lymph recirculation dynamics facilitate a trade-off, and are consistent with dominant roles for the very early detection of rare foreign antigens in a dLN, and the efficient accumulation of signals from systemically distributed self-antigens.

The Deubiquitylase Otub1 Regulates the Chemotactic Response of Splenic B Cells by Modulating the Stability of the γ-Subunit Gng2.

The ubiquitin proteasome system performs the covalent attachment of lysine 48-linked polyubiquitin chains to substrate proteins, thereby targeting them for degradation, while deubiquitylating enzymes (DUBs) reverse this process. This posttranslational modification regulates key features both of innate and adaptative immunity, including antigen presentation, protein homeostasis and signal transduction. Here we show that loss of one of the most highly expressed DUBs, Otub1, results in changes in murine splenic B cell subsets, leading to a significant increase in marginal zone and transitional B cells and a concomitant decrease in follicular B cells. We demonstrate that Otub1 interacts with the γ-subunit of the heterotrimeric G protein, Gng2, and modulates its ubiquitylation status, thereby controlling Gng2 stability. Proximal mapping of Gng2 revealed an enrichment in partners associated with chemokine signaling, actin cytoskeleton and cell migration. In line with these findings, we show that Otub1-deficient B cells exhibit greater Ca2+ mobilization, F-actin polymerization and chemotactic responsiveness to Cxcl12, Cxcl13 and S1P in vitro, which manifests in vivo as altered localization of B cells within the spleen. Together, our data establishes Otub1 as a novel regulator of G-protein coupled receptor signaling in B cells, regulating their differentiation and positioning in the spleen.

Distinctive TLR7 signaling, type I IFN production, and attenuated innate and adaptive immune responses to yellow fever virus in a primate reservoir host.

Why cross-species transmissions of zoonotic viral infections to humans are frequently associated with severe disease when viruses responsible for many zoonotic diseases appear to cause only benign infections in their reservoir hosts is unclear. Sooty mangabeys (SMs), a reservoir host for SIV, do not develop disease following SIV infection, unlike nonnatural HIV-infected human or SIV-infected rhesus macaque (RM) hosts. SIV infections of SMs are characterized by an absence of chronic immune activation, in association with significantly reduced IFN-α production by plasmacytoid dendritic cells (pDCs) following exposure to SIV or other defined TLR7 or TLR9 ligands. In this study, we demonstrate that SM pDCs produce significantly less IFN-α following ex vivo exposure to the live attenuated yellow fever virus 17D strain vaccine, a virus that we show is also recognized by TLR7, than do RM or human pDCs. Furthermore, in contrast to RMs, SMs mount limited activation of innate immune responses and adaptive T cell proliferative responses, along with only transient antiviral Ab responses, following infection with yellow fever vaccine 17D strain. However, SMs do raise significant and durable cellular and humoral immune responses comparable to those seen in RMs when infected with modified vaccinia Ankara, a virus whose immunogenicity does not require TLR7/9 recognition. Hence, differences in the pattern of TLR7 signaling and type I IFN production by pDCs between primate species play an important role in determining their ability to mount and maintain innate and adaptive immune responses to specific viruses, and they may also contribute to determining whether disease follows infection.