TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS-CoV-2 infection.

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID-19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL-6. Using an in vitro stem cell-based human pDC model, we further demonstrate that pDCs, while not supporting SARS-CoV-2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL-6, IL-8, CXCL10) cytokines that protect epithelial cells from de novo SARS-CoV-2 infection. Via targeted deletion of virus-recognition innate immune pathways, we identify TLR7-MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll-like receptor (TLR)2 is responsible for the inflammatory IL-6 response. We further show that SARS-CoV-2 engages the receptor neuropilin-1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL-6 response, suggesting neuropilin-1 as potential therapeutic target for stimulation of TLR7-mediated antiviral protection.

Neuropilin-1 mediates lung tissue-specific control of ILC2 function in type 2 immunity.

Group 2 innate lymphoid cells (ILC2s) are highly heterogeneous tissue-resident lymphocytes that regulate inflammation and tissue homeostasis in health and disease. However, how these cells integrate into the tissue microenvironment to perform tissue-specific functions is unclear. Here, we show neuropilin-1 (Nrp1), which is induced postnatally and sustained by lung-derived transforming growth factor beta-1 (TGFß1), is a tissue-specific marker of lung ILC2s. Genetic ablation or pharmacological inhibition of Nrp1 suppresses IL-5 and IL-13 production by ILC2s and protects mice from the development of pulmonary fibrosis. Mechanistically, TGFß1-Nrp1 signaling enhances ILC2 function and type 2 immunity by upregulating IL-33 receptor ST2 expression. These findings identify Nrp1 as a tissue-specific regulator of lung-resident ILC2s and highlight Nrp1 as a potential therapeutic target for pulmonary fibrosis.

Perspectives and potential approaches for targeting neuropilin 1 in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel type b coronavirus responsible for the COVID-19 pandemic. With over 224 million confirmed infections with this virus and more than 4.6 million people dead because of it, it is critically important to define the immunological processes occurring in the human response to this virus and pathogenetic mechanisms of its deadly manifestation. This perspective focuses on the contribution of the recently discovered interaction of SARS-CoV-2 Spike protein with neuropilin 1 (NRP1) receptor, NRP1 as a virus entry receptor for SARS-CoV-2, its role in different physiologic and pathologic conditions, and the potential to target the Spike-NRP1 interaction to combat virus infectivity and severe disease manifestations.

Unstable regulatory T cells, enriched for naïve and Nrp1neg cells, are purged after fate challenge.

Regulatory T cells (Tregs) are indispensable for the control of immune homeostasis and have clinical potential as a cell therapy for treating autoimmunity. Tregs can lose expression of the lineage-defining Foxp3 transcription factor and acquire effector T cell (Teff) characteristics, a process referred to as Treg plasticity. The extent and reversibility of such plasticity during immune responses remain unknown. Here, using a murine genetic fate-mapping system, we show that Treg stability is maintained even during exposure to a complex microbial/antigenic environment. Furthermore, we demonstrate that the observed plasticity of Tregs after adoptive transfer into a lymphopenic environment is a property limited to only a subset of the Treg population, with the nonconverting majority of Tregs being resistant to plasticity upon secondary stability challenge. The unstable Treg fraction is a complex mixture of phenotypically distinct Tregs, enriched for naïve and neuropilin-1-negative Tregs, and includes peripherally induced Tregs and recent thymic emigrant Tregs These results suggest that a "purging" process can be used to purify stable Tregs that are capable of robust fate retention, with potential implications for improving cell transfer therapy.

Myeloma-Secreted Galectin-1 Potently Interacts with CD304 on Monocytic Myeloid-Derived Suppressor Cells.

Progression of multiple myeloma is regulated by factors intrinsic to the clonal plasma cells (PC) and by the immune effector cells in the tumor microenvironment. In this study, we investigated the interaction between CD304 expression on myeloid-derived suppressor cells (MDSC) and galectin-1 from malignant PCs in the context of autologous stem cell transplantation (ASCT) for multiple myeloma. Using high-throughput screening, CD304 expression on circulating monocytic MDSCs (M-MDSC; CD14+HLA-DRlow/-) was compared before and after ASCT. There was a significantly higher M-MDSC expression of CD304 before ASCT and a clear correlation between circulating pre-ASCT M-MDSC frequency and serum galectin-1 concentration. Treatment of pre-ASCT M-MDSCs, but not post-ASCT M-MDSCs, with galectin-1 in vitro expanded the M-MDSC population and increased expression of CD304. High galectin-1 expression by malignant PCs was associated with poor clinical outcomes. M-MDSC development and expression of CD304 were differentially induced when healthy donor peripheral blood mononuclear cells were cultured with the human multiple myeloma cell lines RPMI-8226 and JJN3, which express high and low galectin-1, respectively. Inhibition of galectin-1 reduced M-MDSC proliferation induced by RPMI-8226 cells but not by JJN3 cells, and blockade of CD304 reduced M-MDSC migration induced by RPMI-8226 cells but not by JJN3 cells. In addition, blockade of CD304 reversed suppression of the in vitro cytotoxic effect of melphalan by pre-ASCT M-MDSCs. Our data demonstrate that multiple myeloma-derived galectin-1 could mediate the tumor-promoting effect of M-MDSCs through its interaction with CD304 on M-MDSCs and contribute to multiple myeloma progression after ASCT.See related Spotlight on p. 488.

Inhibition of neovascularisation in human endothelial cells using anti NRP-1 nanobody fused to truncated form of diphtheria toxin as a novel immunotoxin.

Neuropilin-1 (NRP-1) regulates a range of physiological and pathological processes, including angiogenesis. Targeting of NRP1 is considered a significant approach in cancer therapy. In the present study, a novel antiNRP1 immunotoxin (αNRP1 IT) was developed by genetic fusion of a single domain (VHH) anti-NRP-1 antibody fragment to a truncated diphtheria toxin. The αNRP1 IT was expressed into bacterial cells as an inclusion body (IB). Expression of αNRP1 IT was confirmed by SDS-PAGE and western blotting. Recombinant αNRP1 IT was purified using nickel affinity chromatography. Toxicity and antiangiogenesis effect of αNRP1 IT was investigated both in vitro and in vivo. Results showed that αNRP1 IT significantly reduced the viability of human umbilical vein endothelial cell line (HUVEC) (p < .05). The αNRP1 IT significantly inhibited tube formation of HUVEC cells (p < .001). Furthermore, αNRP1 IT inhibited angiogenesis in Chick Chorioallantoic Membrane (CAM) Assay. These data suggest the potential of αNRP1 IT as a novel therapeutic in targeted cancer therapy.

Can SARS-CoV-2 Virus Use Multiple Receptors to Enter Host Cells?

The occurrence of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVD-19), represents a catastrophic threat to global health. Protruding from the viral surface is a densely glycosylated spike (S) protein, which engages angiotensin-converting enzyme 2 (ACE2) to mediate host cell entry. However, studies have reported viral susceptibility in intra- and extrapulmonary immune and non-immune cells lacking ACE2, suggesting that the S protein may exploit additional receptors for infection. Studies have demonstrated interactions between S protein and innate immune system, including C-lectin type receptors (CLR), toll-like receptors (TLR) and neuropilin-1 (NRP1), and the non-immune receptor glucose regulated protein 78 (GRP78). Recognition of carbohydrate moieties clustered on the surface of the S protein may drive receptor-dependent internalization, accentuate severe immunopathological inflammation, and allow for systemic spread of infection, independent of ACE2. Furthermore, targeting TLRs, CLRs, and other receptors (Ezrin and dipeptidyl peptidase-4) that do not directly engage SARS-CoV-2 S protein, but may contribute to augmented anti-viral immunity and viral clearance, may represent therapeutic targets against COVID-19.

The role of Neuropilin-1 in COVID-19.

Neuropilin-1 (NRP-1), a member of a family of signaling proteins, was shown to serve as an entry factor and potentiate SARS Coronavirus 2 (SARS-CoV-2) infectivity in vitro. This cell surface receptor with its disseminated expression is important in angiogenesis, tumor progression, viral entry, axonal guidance, and immune function. NRP-1 is implicated in several aspects of a SARS-CoV-2 infection including possible spread through the olfactory bulb and into the central nervous system and increased NRP-1 RNA expression in lungs of severe Coronavirus Disease 2019 (COVID-19). Up-regulation of NRP-1 protein in diabetic kidney cells hint at its importance in a population at risk of severe COVID-19. Involvement of NRP-1 in immune function is compelling, given the role of an exaggerated immune response in disease severity and deaths due to COVID-19. NRP-1 has been suggested to be an immune checkpoint of T cell memory. It is unknown whether involvement and up-regulation of NRP-1 in COVID-19 may translate into disease outcome and long-term consequences, including possible immune dysfunction. It is prudent to further research NRP-1 and its possibility of serving as a therapeutic target in SARS-CoV-2 infections. We anticipate that widespread expression, abundance in the respiratory and olfactory epithelium, and the functionalities of NRP-1 factor into the multiple systemic effects of COVID-19 and challenges we face in management of disease and potential long-term sequelae.

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.

Selection and characterization of specific nanobody against neuropilin-1 for inhibition of angiogenesis.

Neuropilin-1 (NRP-1), non-tyrosine kinase receptor, was initially identified as axonal protein and later recognized as co-receptor for vascular endothelial growth factor (VEGF). Neuropilins (NRPs) are involved in vascular development and tumor angiogenesis. Over the last years, many studies have been accomplished to inhibit angiogenesis. In this study, the nanobody library was panned against immobilized NRP-1 antigen. High affinity and specificity nanobodies were selected through monoclonal ELISA. The selected nanobodies inhibited proliferation and tube formation of HUVEC and MCF-7 cells in vitro and ex vivo. The results highlight potential of anti-NRP1 nanobodies in inhibition of angiogenesis both in vitro and ex vivo and promises development of novel therapeutics against pathologic angiogenesis.

Neuropilin-1: a checkpoint target with unique implications for cancer immunology and immunotherapy.

Checkpoint blockade immunotherapy established a new paradigm in cancer treatment: for certain patients curative treatment requires immune reinvigoration. Despite this monumental advance, only 20%-30% of patients achieve an objective response to standard of care immunotherapy, necessitating the consideration of alternative targets. Optimal strategies will not only stimulate CD8+ T cells, but concomitantly modulate immunosuppressive cells in the tumor microenvironment (TME), most notably regulatory T cells (Treg cells). In this context, the immunoregulatory receptor Neuropilin-1 (NRP1) is garnering renewed attention as it reinforces intratumoral Treg cell function amidst inflammation in the TME. Loss of NRP1 on Treg cells in mouse models restores antitumor immunity without sacrificing peripheral tolerance. Enrichment of NRP1+ Treg cells is observed in patients across multiple malignancies with cancer, both intratumorally and in peripheral sites. Thus, targeting NRP1 may safely undermine intratumoral Treg cell fitness, permitting enhanced inflammatory responses with existing immunotherapies. Furthermore, NRP1 has been recently found to modulate tumor-specific CD8+ T cell responses. Emerging data suggest that NRP1 restricts CD8+ T cell reinvigoration in response to checkpoint inhibitors, and more importantly, acts as a barrier to the long-term durability of CD8+ T cell-mediated tumor immunosurveillance. These novel and distinct regulatory mechanisms present an exciting therapeutic opportunity. This review will discuss the growing literature on NRP1-mediated immune modulation which provides a strong rationale for categorizing NRP1 as both a key checkpoint in the TME as well as an immunotherapeutic target with promise either alone or in combination with current standard of care therapeutic regimens.

A Neuropilin-1 Antagonist Exerts Antitumor Immunity by Inhibiting the Suppressive Function of Intratumoral Regulatory T Cells.

Regulatory T cells (Treg) are targeted for cancer immunotherapy because they suppress antitumor immunity. Although the importance of neuropilin-1 (NRP1) in the stability and function of intratumoral Tregs is well-documented, targeting of NRP1+ Tregs for anticancer immunotherapy has not been well explored. Here, we found that an NRP1 antagonist [Fc(AAG)-TPP11], generated by fusion of the NRP1-specific binding peptide TPP11 with the C-terminus of an effector function-deficient immunoglobulin Fc(AAG) variant, inhibits intratumoral NRP1+ Treg function and stability. Fc(AAG)-TPP11 triggered the internalization of NRP1, reducing its surface expression on Tregs and thereby inhibiting the suppressive function of Tregs. In two murine syngeneic tumor models, Fc(AAG)-TPP11 retarded tumor growth, comparable with a Treg-depleting anti-CTLA-4 antibody, without noticeable toxicity. Fc(AAG)-TPP11 inhibited NRP1-dependent Treg function, inducing unstable intratumoral Tregs, with reduced expression of Foxp3 and enhanced production of IFNγ, which subsequently increased the functionality and frequency of intratumoral CD8+ T cells. We also observed selective expression of NRP1 on Tregs isolated from human tumors, but not from the blood of healthy donors and patients with cancer, as well as ex vivo inhibition of intratumoral NRP1+ Treg function by Fc(AAG)-TPP11. Our results suggest that the NRP1 antagonist Fc(AAG)-TPP11 has therapeutic potential for the inhibition of intratumoral NRP1+ Tregs with limited unfavorable effects on peripheral Tregs.

Class-switch recombination to IgA in the Peyer's patches requires natural thymus-derived Tregs and appears to be antigen independent.

Our understanding of how class-switch recombination (CSR) to IgA occurs in the gut is still incomplete. Earlier studies have indicated that Tregs are important for IgA CSR and these cells were thought to transform into follicular helper T cells (Tfh), responsible for germinal center formation in the Peyer's patches (PP). Following adoptive transfer of T-cell receptor-transgenic (TCR-Tg) CD4 T cells into nude mice, we unexpectedly found that oral immunization did not require an adjuvant to induce strong gut IgA and systemic IgG responses, suggesting an altered regulatory environment in the PP. After sorting of splenic TCR-Tg CD4 T cells into CD25+ or CD25- cells we observed that none of these fractions supported a gut IgA response, while IgG responses were unperturbed in mice receiving the CD25- cell fraction. Hence, while Tfh functions resided in the CD25- fraction the IgA CSR function in the PP was dependent on CD25+ Foxp3+ Tregs, which were found to be Helios+ neuropilin-1+ thymus-derived Tregs. This is the first study to demonstrate that Tfh and IgA CSR functions are indeed, unique, and separate functions in the PP with the former being TCR-dependent while the latter appeared to be antigen independent.

Construction and characterization of a truncated tissue factor­coagulation­based composite system for selective thrombosis in tumor blood vessels.

The selective induction of tumor vascular thrombosis using truncated tissue factor (tTF) delivered via a target ligand is a promising novel antitumor strategy. In the present study, an anti­neuropilin­1 (NRP­1) monoclonal antibody (mAb)­streptavidin (SA):tTF­biotin (B) composite system was established. In this system, anti­NRP­1­mAb located tTF to the tumor vascular endothelial cell surface and induced vascular embolization. Due to their high binding affinity, SA and B were used to enhance thrombogenic activity. mAb was conjugated with SA using a coupling method with water­soluble 1­ethyl­3­(3­dimethylaminopropyl) carbodiimide and N­hydroxysulfosuccinimide. Biotinylated tTF (tTF­B) was prepared using a B­labeling kit subsequent to the generation and purification of fusion protein tTF. Confocal microscopy and flow cytometry indicated that the anti­NRP­1­mAb­SA conjugate retained mAb targeting activity. The preservation of B­conjugate binding capacity was confirmed using a competitive ELISA, and factor X­activation analysis revealed that tTF­B retained the procoagulant activity exhibited by tTF. Live imaging was performed to assess mAb­SA distribution and tumor­targeting capability, and this yielded promising results. The results of in vivo studies in mice with subcutaneous xenografts demonstrated that this composite system significantly induced tumor vascular thrombosis and inhibited tumor growth, whereas these histological changes were not observed in normal organs.

Daurinol Attenuates Autoimmune Arthritis via Stabilization of Nrp1-PTEN-Foxp3 Signaling in Regulatory T Cells.

Optimizing Treg function and improving Treg stability are attractive treatment strategies for treating autoimmune rheumatoid arthritis (RA). However, the limited number of circulating Tregs and questions about the functional stability of in vitro-expanded Tregs are potential limitations of Treg-based cell therapy. The aim of this study was to analyze the regulatory effect of daurinol, a catalytic inhibitor of topoisomerase IIα, on Th cell differentiation and to evaluate their therapeutic potential in a preclinical experimental model of RA. We investigated the effect of daurinol on T cell differentiation by flow cytometry. Foxp3 stability and methylation were analyzed by suppression assays and bisulfite pyrosequencing. Daurinol was treated in the collagen-induced arthritis (CIA) model, and the effects in vivo were determined. We found that daurinol can promote Treg differentiation and reciprocally inhibit Th17 differentiation. This Treg-inducing property of daurinol was associated with decreased activity of Akt-mTOR and reciprocally increased activity of neuropilin-1 (Nrp1)-PTEN. Daurinol treatment inhibited aerobic glycolysis in Th17 conditions, indicating the metabolic changes by daurinol. We found that the daurinol increase the Treg stability was achieved by Foxp3 hypomethylation. In vivo daurinol treatment in CIA mice reduced the clinical arthritis severity and histological inflammation. The Treg population frequency increased and the Th17 cells decreased in the spleens of arthritis mice treated with daurinol. These results showed the anti-arthritic and immunoregulating properties of daurinol is achieved by increased differentiation and stabilization of Tregs. Our study provides first evidence for daurinol as a treatment for RA.

Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1.

Neuropilin-1 (Nrp-1) is a marker for murine CD4+FoxP3+ regulatory T (Treg) cells, a subset of human CD4+ Treg cells, and a population of CD8+ T cells infiltrating certain solid tumours. However, whether Nrp-1 regulates tumour-specific CD8 T-cell responses is still unclear. Here we show that Nrp-1 defines a subset of CD8+ T cells displaying PD-1hi status and infiltrating human lung cancer. Interaction of Nrp-1 with its ligand semaphorin-3A inhibits migration and tumour-specific lytic function of cytotoxic T lymphocytes. In vivo, Nrp-1+PD-1hi CD8+ tumour-infiltrating lymphocytes (TIL) in B16F10 melanoma are enriched for tumour-reactive T cells exhibiting an exhausted state, expressing Tim-3, LAG-3 and CTLA-4 inhibitory receptors. Anti-Nrp-1 neutralising antibodies enhance the migration and cytotoxicity of Nrp-1+PD-1hi CD8+ TIL ex vivo, while in vivo immunotherapeutic blockade of Nrp-1 synergises with anti-PD-1 to enhance CD8+ T-cell proliferation, cytotoxicity and tumour control. Thus, Nrp-1 could be a target for developing combined immunotherapies.

Inflammatory Responses Reprogram TREGS Through Impairment of Neuropilin-1.

Chronic inflammatory insults compromise immune cell responses and ultimately contribute to pathologic outcomes. Clinically, it has been suggested that bone debris and implant particles, such as polymethylmethacrylate (PMMA), which are persistently released following implant surgery evoke heightened immune, inflammatory, and osteolytic responses that contribute to implant failure. However, the precise mechanism underlying this pathologic response remains vague. TREGS, the chief immune-suppressive cells, express the transcription factor Foxp3 and are potent inhibitors of osteoclasts. Using an intra-tibial injection model, we show that PMMA particles abrogate the osteoclast suppressive function of TREGS. Mechanistically, PMMA particles induce TREG instability evident by reduced expression of Foxp3. Importantly, intra-tibial injection of PMMA initiates an acute innate immune and inflammatory response, yet the negative impact on TREGS by PMMA remains persistent. We further show that PMMA enhance TH17 response at the expense of other T effector cells (TEFF), particularly TH1. At the molecular level, gene expression analysis showed that PMMA particles negatively regulate Nrp-1/Foxo3a axis to induce TREG instability, to dampen TREG activity and to promote phenotypic switch of TREGS to TH17 cells. Taken together, inflammatory cues and danger signals, such as bone and implant particles exacerbate inflammatory osteolysis in part through reprogramming TREGS.

CARD11 is dispensable for homeostatic responses and suppressive activity of peripherally induced FOXP3+ regulatory T cells.

FOXP3+ regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1+ (NRP1+ ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1- Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4+ T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein.

Neuropilin-1 Regulates the Secondary CD8 T Cell Response to Virus Infection.

Neuropilin-1 (Nrp1) plays important roles in axonal guidance in neurons and in the growth of new blood vessels. There is also a growing appreciation for roles played by neuropilin-1 in the immune response. This molecule is important for the function of regulatory T cells; however, roles in other T cell populations have not been identified. Here, we show that neuropilin-1 is expressed during the peak of the antiviral CD8 T cell response during murine gammaherpesvirus infection. Using a conditional knockout model, we deleted Nrp1 either before infection or after CD8 T cell memory had been established. We found that deletion of Nrp1 skewed the acute CD8 T cell response toward a memory precursor-like phenotype; however, the ensuing resting memory response was similar regardless of Nrp1 expression. Interestingly, Nrp1 deletion had differing effects on the recall response depending on the timing of deletion. When deleted before infection, Nrp1 deficiency inhibited the secondary response. Deletion just prior to reexposure to virus led to an enhanced secondary response. Interestingly, these effects were observed only in mice infected with a persistent strain of murine gammaherpesvirus and not with a nonpersistent mutant strain. These data highlight a multifaceted role for neuropilin-1 in memory CD8 T cell differentiation, dependent upon the stage of the T cell response and characteristics of the infectious agent. Several therapeutic anticancer therapies focus on inhibition of Nrp1 to restrict tumor growth, and so knowledge of how Nrp1 blockade may affect the CD8 T cell response will provide a better understanding of treatment consequences.IMPORTANCE CD8 T cell responses are critical to control both virus infections and tumors. The ability of these cells to persist for long periods of time can result in lifelong immunity, as relatively small populations of cells can expand rapidly to counter reexposure to the same insult. Understanding the molecules necessary for this rapid secondary expansion is critical if we are to develop therapies that can provide lifelong protection. This report shows an important and complex role for the molecule neuropilin-1 in the secondary response. Several cancer therapies targeting neuropilin-1 are in development, and this work will lead to better understanding of the effect these therapies could have upon the protective CD8 T cell response.