Vaginal Immunization to Elicit Primary T-Cell Activation and Dissemination

Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4⁺ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4⁺ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs.     

Mast cell activators: a new class of highly effective vaccine adjuvants

Mast cells (MCs) have recently received recognition as prominent effectors in the regulation of immune cell migration to draining lymph nodes and lymphocyte activation. However, their role in the development of humoral immune responses is not clear. Here, we demonstrate that subcutaneous or nasal administration of small-molecule MC activators with vaccine antigens evokes large increases in antigen-specific serum immunoglobulin G (IgG) responses. These responses were MC dependent and correlated with increased dendritic cell and lymphocyte recruitment to draining lymph nodes. Nasal instillation of these formulations also evoked antigen-specific secretory IgA and provided protection against anthrax lethal toxin challenge in vitro and against vaccinia virus infection in vivo. Collectively, these results define the MC as an integral sensory arm of the adaptive immune system. Moreover, they highlight MC activators as a new class of vaccine adjuvants, capable of inducing protective antigen-specific immune responses through needle-free routes of administration.     

Inflammation, lymphatic function, and dendritic cell migration

The lymphatic system is not only essential for maintenance of normal fluid balance, but also for proper immunologic function by providing an extensive network of vessels, important for cell trafficking and antigen delivery, as well as an exclusive environment, the lymph node (LN), where antigen-presenting cells (APCs) and lymphocytes can encounter and interact. Among APCs, dendritic cells (DCs) have a remarkable capacity to traffic from peripheral tissues to the draining LN, which is critical for execution of their functions. To reach the LN, DCs must migrate towards and enter lymphatic vessels. Here, the authors review what is known about the factors that drive this process. They touch particularly on the topic of how DC migration is affected by inflammation and discuss this in the context of lymphatic function. Traditionally, inflammatory mediators are regarded to support DC migration to LNs because they induce molecules on DCs known to guide them to lymphatics. The authors recently showed that inflammatory signals present in a strong vaccine adjuvant induce swelling in LNs accompanied by lymphangiogenesis in the draining LN and radius of peripheral tissue. These increased lymphatics, at least for several days, lead to a more robust migration of DCs. However, the density of lymphatic vessels can become overly extended and/or their function impaired as observed during lymphedema and various chronic inflammatory reactions. Diseases characterized by chronic inflammation often present with impaired DC migration and adaptive immunity. Gaining a better understanding of how lymphatic vessel function may impact adaptive immunity by, for example, altering DC migration will benefit clinical research aiming to manipulate immune responses and manage chronic inflammatory diseases.     

A novel laser vaccine adjuvant increases the motility of antigen presenting cells

Background

Development of a potent vaccine adjuvant without introduction of any side effects remains an unmet challenge in the field of the vaccine research.

Methodology/Principal Findings

We found that laser at a specific setting increased the motility of antigen presenting cells (APCs) and immune responses, with few local or systemic side effects. This laser vaccine adjuvant (LVA) effect was induced by brief illumination of a small area of the skin or muscle with a nondestructive, 532 nm green laser prior to intradermal (i.d.) or intramuscular (i.m.) administration of vaccines at the site of laser illumination. The pre-illumination accelerated the motility of APCs as shown by intravital confocal microscopy, leading to sufficient antigen (Ag)-uptake at the site of vaccine injection and transportation of the Ag-captured APCs to the draining lymph nodes. As a result, the number of Ag⁺ dendritic cells (DCs) in draining lymph nodes was significantly higher in both the 1° and 2° draining lymph nodes in the presence than in the absence of LVA. Laser-mediated increases in the motility and lymphatic transportation of APCs augmented significantly humoral immune responses directed against a model vaccine ovalbumin (OVA) or influenza vaccine i.d. injected in both primary and booster vaccinations as compared to the vaccine itself. Strikingly, when the laser was delivered by a hair-like diffusing optical fiber into muscle, laser illumination greatly boosted not only humoral but also cell-mediated immune responses provoked by i.m. immunization with OVA relative to OVA alone.

Conclusion/Significance

The results demonstrate the ability of this safe LVA to augment both humoral and cell-mediated immune responses. In comparison with all current vaccine adjuvants that are either chemical compounds or biological agents, LVA is novel in both its form and mechanism; it is risk-free and has distinct advantages over traditional vaccine adjuvants.     

Analysis of in vivo immune responses during Toxoplasma gondii infection using the technique of lymphatic cannulation

A major obstacle in the design of effective inactivated vaccines is the induction of an appropriate immune response in the recipient. Direct examination of immune responses induced in vivo following infection is possible using the technique of lymphatic cannulation. This allows a unique insight into the development of the immune response to particular pathogens and provides important information that could be exploited in the design of suitable vaccine delivery systems in order to induce and maintain appropriate protective immune responses. In this article, Elisabeth Innes and Jonathan Wastling review studies involving the use of lymphatic cannulation to examine immune responses induced in vivo during infection of sheep with the S48 strain of Toxoplasma gondii, which is currently used as a vaccine against toxoplasmosis for vetennary use only.     

Distribution of primed T cells and antigen-loaded antigen presenting cells following intranasal immunization in mice

Priming of T cells is a key event in vaccination, since it bears a decisive influence on the type and magnitude of the immune response. T-cell priming after mucosal immunization via the nasal route was studied by investigating the distribution of antigen-loaded antigen presenting cells (APCs) and primed antigen-specific T cells. Nasal immunization studies were conducted using the model protein antigen ovalbumin (OVA) plus CpG oligodeoxynucleotide adjuvant. Trafficking of antigen-specific primed T cells was analyzed in vivo after adoptive transfer of OVA-specific transgenic T cells in the presence or absence of fingolimod, a drug that causes lymphocytes sequestration within lymph nodes. Antigen-loaded APCs were observed in mediastinal lymph nodes, draining the respiratory tract, but not in distal lymph nodes. Antigen-specific proliferating T cells were first observed within draining lymph nodes, and later in distal iliac and mesenteric lymph nodes and in the spleen. The presence at distal sites was due to migration of locally primed T cells as shown by fingolimod treatment that caused a drastic reduction of proliferated T cells in non-draining lymph nodes and an accumulation of extensively divided T cells within draining lymph nodes. Homing of nasally primed T cells in distal iliac lymph nodes was CD62L-dependent, while entry into mesenteric lymph nodes depended on both CD62L and α4β7, as shown by in vivo antibody-mediated inhibition of T-cell trafficking. These data, elucidating the trafficking of antigen-specific primed T cells to non-draining peripheral and mucosa-associated lymph nodes following nasal immunization, provide relevant insights for the design of vaccination strategies based on mucosal priming.     

Isolation and purification of afferent lymph dendritic cells that drain the skin of cattle

Dendritic cells (DCs) are central to the induction of immune responses and are a pivotal control point that determines the outcome of infectious challenge. Cannulation of afferent lymphatic vessels allows the isolation of large numbers of lymph DCs. First, lymph nodes that are draining the skin are surgically removed (takes approximately 1 h). Over a period of 6-8 weeks, afferent lymphatic vessels re-anastomose with the efferent duct, forming larger 'pseudoafferent' lymphatic vessels that can be surgically cannulated. Surgical cannulation takes 2 h to perform; daily maintenance of the catheter requires 30 min. Isolation of lymph cells requires 1 h and an additional 60-180 min to enrich or purify the DCs. The lymph can be harvested for up to 1 month, with relatively constant cell numbers and subset distribution throughout this period. This technique, although technically demanding, facilitates studies of DCs and other cells that traffic in the lymph in both the steady state and following antigenic exposure.     

Human lymphatic endothelial cells express multiple functional TLRs

The lymphatic endothelium is the preferred route for the drainage of interstitial fluid from tissues and also serves as a conduit for peripheral dendritic cells (DCs) to reach draining lymph nodes. Lymphatic endothelial cells (LECs) are known to produce chemokines that recruit Ag-loaded DCs to lymphatic vessels and therefore are likely to regulate the migration of DCs to lymph nodes. TLRs are immune receptors that recognize pathogen associated molecular patterns and then signal and stimulate production of inflammatory chemokines and cytokines that contribute to innate and adaptive immune responses. TLRs are known to be expressed by a wide variety of cell types including leukocytes, epithelial cells, and endothelial cells. Because the TLR expression profile of LECs remains largely unexamined, we have undertaken a comprehensive study of the expression of TLR1-10 mRNAs and protein in primary human dermal (HD) and lung LECs as well as in htert-HDLECs, which display a longer life-span than HDLECs. We found that all three cell types expressed TLR1-6 and TLR9. The responsiveness of these LECs to a panel of ligands for TLR1-9 was measured by real-time RT-PCR, ELISA, and flow cytometry, and revealed that the LECs responded to most but not all TLR ligands by increasing expression of inflammatory chemokines, cytokines, and adhesion molecules. These findings provide insight into the ability of cells of the lymphatic vasculature to respond to pathogens and potential vaccine adjuvants and shape peripheral environments in which DCs will acquire Ag and environmental cues.     

Mucosally delivered dendritic cells activate T cells independently of IL-12 and endogenous APCs

In vitro manipulated dendritic cells (DC) have increasingly been used as a promising vaccine formulation against cancer and infectious disease. However, improved understanding of the immune mechanisms is needed for the development of safe and efficacious mucosal DC immunization. We have developed a murine model of respiratory mucosal immunization by using a genetically manipulated DC vaccine. Within 24 h of intranasal delivery, the majority of vaccine DCs migrated to the lung mucosa and draining lymph nodes and elicited a significant level of T cells capable of IFN-gamma secretion and CTL in the airway lumen as well as substantial T cell responses in the spleen. And such T cell responses were associated with enhanced protection against respiratory mucosal intracellular bacterial challenge. In comparison, parenteral i.m. DC immunization did not elicit marked airway luminal T cell responses and immune protection regardless of strong systemic T cell activation. Although repeated mucosal DC delivery boosted Ag-specific T cells in the airway lumen, added benefits to CD8 T cell activation and immune protection were not observed. By using MHC-deficient vaccine DCs, we further demonstrated that mucosal DC immunization-mediated CD8 and CD4 T cell activation does not require endogenous DCs. By using IL-12-deficient vaccine DCs, we also observed that IL-12(-/-) DCs failed to migrate to the lymph nodes but remained capable of T cell activation. Our observations indicate that mucosal delivery of vaccine DCs represents an effective approach to enhance mucosal T cell immunity, which may operate independent of vaccine IL-12 and endogenous DCs.     

Enhanced lymph node retention of subcutaneously injected IgG1-PEG2000-liposomes through pentameric IgM antibody-mediated vesicular aggregation

An efficient strategy for enhancing the lymph node deposition of rapidly drained liposomes from the interstitial injection site is described. Subcutaneously injected small-sized immuno-poly(ethyleneglycol)-liposomes (immuno-PEG-liposomes), containing 10 mol% mPEG350-phospholipid and 1 mol% PEG2000-phospholipid in their bilayer and where IgG1 is coupled to the distal end of PEG2000, not only drain rapidly from the interstitial spaces into the initial lymphatic system, but also accumulate efficiently among the lymph nodes draining the region when compared with non-PEG-bearing immunoliposomes where IgG is directly coupled to the phospholipid. Liposome deposition among the draining lymph nodes, however, was further enhanced dramatically following an adjacent subcutaneous injection of a pentameric IgM against the surface attached IgG molecules (IgM:IgG, 10:1) without compromising vesicle drainage from the interstitium. This is suggested to arise either as a result of formation of large immuno-aggregates within the lymphatic vessels with subsequent transport to and trapping among the regional lymph nodes and/or following IgM binding to Fc receptors of the lymph node sinus macrophages forming a platform for subsequent trapping of drained IgG-coupled liposomes. This lymph node targeting approach may be amenable for the design and surface engineering of any rapidly drained nanoparticulate system bearing peptides and proteins that can be aggregated with a desired monoclonal pentameric IgM.     

Dendritic-cell trafficking to lymph nodes through lymphatic vessels

Antigen-presenting dendritic cells often acquire foreign antigens in peripheral tissues such as the skin. Optimal encounter with naive T cells for the presentation of these antigens requires that the dendritic cells migrate to draining lymph nodes through lymphatic vessels. In this article, we review important aspects of what is known about dendritic-cell trafficking into and through lymphatic vessels to lymph nodes. We present these findings in the context of information about lymphatic-vessel biology. Gaining a better understanding of the crosstalk between dendritic cells and lymphatic vessels during the migration of dendritic cells to lymph nodes is essential for future advances in manipulating dendritic-cell migration as a means to fine-tune immune responses in clinical settings.     

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis BCG: anatomical sites of bacterial replication and immune activity

Lipid microencapsulation of Mycobacterium bovis bacille Calmette-Guérin (BCG) produces an oral delivery vaccine that can establish systemic cell-mediated immune reactivity and protection against aerosol mycobacterial challenge in mice. Here, we describe the lymphatic and mucosal sites of bacterial replication, and location of Mycobacterium-specific IFN-gamma-secreting cell populations, following oral vaccination of BALB/c mice. Eight weeks following a single oral dose of lipid-encapsulated BCG, viable BCG organisms were recovered from the mesenteric lymph nodes (MLN) of 11/12 mice investigated (93%). Live bacteria were also occasionally recovered from the cervical lymph nodes (17%) and Peyer's patches (8%), but not from homogenates of the lungs or spleen. Strong Mycobacterium-specific IFN-gamma production was recorded among isolated splenocytes, but not among populations of mononuclear cells derived from the MLN or lungs. Oral vaccination of mice with lipid-encapsulated BCG thus appears to promote a state of systemic immunological reactivity more akin to that observed following parenteral rather than conventional oral vaccination, despite the fact that replicating bacilli are restricted to lymphatic tissues of the alimentary tract. Possible patterns of lymphocyte sensitization and trafficking are discussed.     

Enhanced lymph node retention of subcutaneously injected IgG1-PEG2000-liposomes through pentameric IgM antibody-mediated vesicular aggregation

An efficient strategy for enhancing the lymph node deposition of rapidly drained liposomes from the interstitial injection site is described. Subcutaneously injected small-sized immuno-poly(ethyleneglycol)-liposomes (immuno-PEG-liposomes), containing 10 mol% mPEG₃₅₀-phospholipid and 1 mol% PEG₂₀₀₀-phospholipid in their bilayer and where IgG1 is coupled to the distal end of PEG₂₀₀₀, not only drain rapidly from the interstitial spaces into the initial lymphatic system, but also accumulate efficiently among the lymph nodes draining the region when compared with non-PEG-bearing immunoliposomes where IgG is directly coupled to the phospholipid. Liposome deposition among the draining lymph nodes, however, was further enhanced dramatically following an adjacent subcutaneous injection of a pentameric IgM against the surface attached IgG molecules (IgM:IgG, 10:1) without compromising vesicle drainage from the interstitium. This is suggested to arise either as a result of formation of large immuno-aggregates within the lymphatic vessels with subsequent transport to and trapping among the regional lymph nodes and/or following IgM binding to Fc receptors of the lymph node sinus macrophages forming a platform for subsequent trapping of drained IgG-coupled liposomes. This lymph node targeting approach may be amenable for the design and surface engineering of any rapidly drained nanoparticulate system bearing peptides and proteins that can be aggregated with a desired monoclonal pentameric IgM.     

Exploiting lymphatic transport and complement activation in nanoparticle vaccines

Antigen targeting and adjuvancy schemes that respectively facilitate delivery of antigen to dendritic cells and elicit their activation have been explored in vaccine development. Here we investigate whether nanoparticles can be used as a vaccine platform by targeting lymph node-residing dendritic cells via interstitial flow and activating these cells by in situ complement activation. After intradermal injection, interstitial flow transported ultra-small nanoparticles (25 nm) highly efficiently into lymphatic capillaries and their draining lymph nodes, targeting half of the lymph node-residing dendritic cells, whereas 100-nm nanoparticles were only 10% as efficient. The surface chemistry of these nanoparticles activated the complement cascade, generating a danger signal in situ and potently activating dendritic cells. Using nanoparticles conjugated to the model antigen ovalbumin, we demonstrate generation of humoral and cellular immunity in mice in a size- and complement-dependent manner.     

Cytokine requirements for induction of systemic and mucosal CTL after nasal immunization.

Cholera toxin (CT) is frequently used as an experimental adjuvant intranasally for the induction of systemic and mucosal immunity. However, CT is highly reactogenic and not approved for use in humans. To define the cytokine requirements for the nasal activation of the systemic and mucosal immune system, and to design new adjuvants with efficacy similar to CT, we defined the cytokines that were able to replace CT as a nasal adjuvant for the induction of CTL. BALB/c mice were nasally immunized with an HIV immunogen that contains an MHC class I-restricted CTL epitope +/- cytokines and tested for HIV-specific immune responses. We found that combinations of IL-1alpha plus IL-18, IL-1alpha plus IL-12, and IL-1alpha plus IL-12 plus GM-CSF each induced optimal splenocyte anti-HIV CTL responses in immunized mice (range 60-71% peptide-specific (51)Cr release). Peak H-2D(d)-peptide tetramer-binding T cell responses induced by cytokine combinations were up to 5.5% of CD8(+) PBMC. Nasal immunization with HIV immunogen and IL-1alpha, IL-12, and GM-CSF also induced Ag-specific IFN-gamma-secreting cells in the draining cervical lymph node and the lung. The use of IL-1alpha, IL-12, and GM-CSF as nasal adjuvants was associated with an increased expression of MHC class II and B7.1 on nonlymphocytes within the nasal-associated lymphoid tissue/nasal mucosa. Thus, IL-1alpha, IL-12, IL-18, and GM-CSF are critical cytokines for the induction of systemic and mucosal CTL after nasal immunization. Moreover, these cytokines may serve as effective adjuvants for nasal vaccine delivery.     

Antigen presentation by exosomes released from peptide-pulsed dendritic cells is not suppressed by the presence of active CTL

Despite the potency of dendritic cells (DCs) as a vaccine carrier, they are short-lived and sensitive to CTL-mediated elimination. Thus, it is believed that the longevity of Ag presentation by peptide-pulsed DC is limited in vivo. Surprisingly, however, we found that although the majority of injected DCs disappeared from the draining lymph nodes within 7 days, Ag presentation persisted for at least 14 days following DC immunization. This prolonged Ag presentation was not mediated by the remaining injected DCs or through Ag transfer to endogenous APCs. We provide evidence that exosomes released by DCs might be responsible for the persistence of Ag presentation. Functional exosomes could be recovered from the draining lymph nodes of C57BL/6 mice following DC vaccination and, in contrast to DCs, T cell stimulation by exosomes in vivo was not affected by the presence of CTL. Our findings demonstrate that Ag presentation following delivery of DC vaccines persists for longer than expected and indicate that the exosome may play a previously unrecognized role in Ag presentation following DC vaccination. Furthermore, our study reinforces the application of exosomes as a vaccination platform and suggests that exosome-based vaccines may be advantageous for booster immunizations due to their resistance to CTL.     

APCs in the anterior uveal tract do not migrate to draining lymph nodes

The migration of APCs from sites of infection and their maturation are critical elements in the generation of immune responses. However, the paths by which intraocular Ags migrate to draining lymph nodes are not known because the eye has limited lymphatic vessels. To date, only dendritic cells from the cornea and conjunctiva have been shown to emigrate. We demonstrate that phagocytic APCs in the anterior uveal tissues of the murine eye that ingest fluorescent latex beads do not migrate to regional lymph nodes. The beads are ingested in the uveal tract by cells expressing MHC class II, CD11c, or F4/80. Using intravital time-lapse videomicroscopy to monitor iris APC migration after anterior chamber injection of fluorescent Ag, fluorescently labeled APCs fail to move at multiple observation times, even in the presence of Ag and LPS. Whereas an as yet unidentified ocular nonphagocytic APC subset might migrate from the anterior uveal tissues, it is more probable that immune responses in the draining lymph nodes are engendered by soluble Ag escaping the eye through interstitial spaces. The inability of anterior uveal tissue APCs to migrate to lymph nodes may contribute to deviant immune responses that dominate after Ags are introduced into the anterior chamber.     

The Effect of Plant Tissue and Vaccine Formulation on the Oral Immunogenicity of a Model Plant-Made Antigen in Sheep

Antigen-specific antibody responses against a model antigen (the B subunit of the heat labile toxin of enterotoxigenic Escherichia coli, LTB) were studied in sheep following oral immunisation with plant-made and delivered vaccines. Delivery from a root-based vehicle resulted in antigen-specific immune responses in mucosal secretions of the abomasum and small intestine and mesenteric lymph nodes. Immune responses from the corresponding leaf-based vaccine were more robust and included stimulation of antigen-specific antibodies in mucosal secretions of the abomasum. These findings suggest that oral delivery of a plant bioencapsulated antigen can survive passage through the rumen to elicit mucosal and systemic immune responses in sheep. Moreover, the plant tissue used as the vaccine delivery vehicle affects the magnitude of these responses.     

Rapid Transepithelial Transport of Prions following Inhalation

Prion infection and pathogenesis are dependent on the agent crossing an epithelial barrier to gain access to the recipient nervous system. Several routes of infection have been identified, but the mechanism(s) and timing of in vivo prion transport across an epithelium have not been determined. The hamster model of nasal cavity infection was used to determine the temporal and spatial parameters of prion-infected brain homogenate uptake following inhalation and to test the hypothesis that prions cross the nasal mucosa via M cells. A small drop of infected or uninfected brain homogenate was placed below each nostril, where it was immediately inhaled into the nasal cavity. Regularly spaced tissue sections through the entire extent of the nasal cavity were processed immunohistochemically to identify brain homogenate and the disease-associated isoform of the prion protein (PrP^d). Infected or uninfected brain homogenate was identified adhering to M cells, passing between cells of the nasal mucosa, and within lymphatic vessels of the nasal cavity at all time points examined. PrP^d was identified within a limited number of M cells 15 to 180 min following inoculation, but not in the adjacent nasal mucosa-associated lymphoid tissue (NALT). While these results support M cell transport of prions, larger amounts of infected brain homogenate were transported paracellularly across the respiratory, olfactory, and follicle-associated epithelia of the nasal cavity. These results indicate that prions can immediately cross the nasal mucosa via multiple routes and quickly enter lymphatics, where they can spread systemically via lymph draining the nasal cavity.     

Increased survival in sepsis by in vivo adenovirus-induced expression of IL-10 in dendritic cells

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (10(5)) yields only local expression, while transduction with higher particle numbers (10(7) and 10(10)) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (10(5)) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.