An Aptamer-Based Proteomic Analysis of Plasma from Cats (Felis catus) with Clinical Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) is a systemic disease manifestation of feline coronavirus (FCoV) and is the most important cause of infectious disease-related deaths in domestic cats. FIP has a variable clinical manifestation but is most often characterized by widespread vasculitis with visceral involvement and/or neurological disease that is typically fatal in the absence of antiviral therapy. Using an aptamer-based proteomics assay, we analyzed the plasma protein profiles of cats who were naturally infected with FIP (n = 19) in comparison to the plasma protein profiles of cats who were clinically healthy and negative for FCoV (n = 17) and cats who were positive for the enteric form of FCoV (n = 9). We identified 442 proteins that were significantly differentiable; in total, 219 increased and 223 decreased in FIP plasma versus clinically healthy cat plasma. Pathway enrichment and associated analyses showed that differentiable proteins were related to immune system processes, including the innate immune response, cytokine signaling, and antigen presentation, as well as apoptosis and vascular integrity. The relevance of these findings is discussed in the context of previous studies. While these results have the potential to inform diagnostic, therapeutic, and preventative investigations, they represent only a first step, and will require further validation.

Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses.

Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.

Review: The PI3K-AKT-mTOR signal transduction pathway in canine cancer.

Tumors in dogs and humans share many similar molecular and genetic features, incentivizing a better understanding of canine neoplasms not only for the purpose of treating companion animals, but also to facilitate research of spontaneously developing tumors with similar biologic behavior and treatment approaches in an immunologically competent animal model. Multiple tumor types of both species have similar dysregulation of signal transduction through phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB; AKT), and mechanistic target of rapamycin (mTOR), collectively known as the PI3K-AKT-mTOR pathway. This review aims to delineate the pertinent aspects of the PI3K-AKT-mTOR signaling pathway in health and in tumor development. It will then present a synopsis of current understanding of PI3K-AKT-mTOR signaling in important canine cancers and advancements in targeted inhibitors of this pathway.

Expression of E. coli FimH Enhances Trafficking of an Orally Delivered Lactobacillus acidophilus Vaccine to Immune Inductive Sites via Antigen-Presenting Cells.

The development of lactic acid bacteria as mucosal vaccine vectors requires the identification of robust mucosal adjuvants to increase vaccine effectiveness. The E. coli type I fimbriae adhesion protein FimH is of interest as a mucosal adjuvant as it targets microfold (M) cells enhancing vaccine uptake into Peyer's patches and can activate the innate immune system via Toll-like receptor (TLR) 4 binding. Here, we displayed the N-terminal domain of FimH on the surface of a Lactobacillus acidophilus vaccine vector and evaluated its ability to increase uptake of L. acidophilus into Peyer's patches and activate innate immune responses. FimH was robustly displayed on the L. acidophilus surface but did not increase uptake into the Peyer's patches. FimH did increase trafficking of L. acidophilus to mesenteric lymph nodes by antigen-presenting cells including macrophages and dendritic cells. It also increased transcription of retinaldehyde dehydrogenase and decreased transcription of IL-21 in the Peyer's patches and mesenteric lymph nodes. The N-terminal domain of FimH did not activate TLR4 in vitro, indicating that FimH may stimulate innate immune responses through a not-yet-identified mechanism. These results indicate that E. coli FimH alters the innate immune response to L. acidophilus and should be further studied as an adjuvant for lactic acid bacterial vaccine platforms.

Oral Vaccination Using a Probiotic Vaccine Platform Combined with Prebiotics Impacts Immune Response and the Microbiome.

Unique to mucosal vaccination is the reciprocal influence of the microbiome and mucosal immune responses, where the immune system is constantly balancing between the clearance of pathogens and the tolerance of self-antigen, food, and the microbiota. Secretory IgA plays a major role in maintaining the homeostasis of a healthy gut microbiome. Natural polyreactive IgA often coats members of the commensal microbiota to aid in their colonization, while high-affinity specific IgA binds to pathogens resulting in their clearance. We developed a probiotic-based mucosal vaccination platform using the bacterium Lactobacillus acidophilus (rLA) with the potential to influence this balance in the IgA coating. In this study, we sought to determine whether repeated administration of rLA alters the host intestinal microbial community due to the immune response against the rLA vaccine. To address this, IgA-seq was employed to characterize shifts in IgA-bound bacterial populations. Additionally, we determined whether using rice bran as a prebiotic would influence the immunogenicity of the vaccine and/or IgA-bound bacterial populations. Our results show that the prebiotic influenced the kinetics of rLA antibody induction and that the rLA platform did not cause lasting disturbances to the microbiome.

High-throughput viral microneutralization method for feline coronavirus using image cytometry.

Feline coronaviruses (FCoV) are members of the alphacoronavirus genus that are further characterized by serotype (types I and II) based on the antigenicity of the spike (S) protein and by pathotype based on the associated clinical conditions. Feline enteric coronaviruses (FECV) are associated with the vast majority of infections and are typically asymptomatic. Within individual animals, FECV can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (FIP), the leading infectious cause of death in domestic cat populations. There are no approved antiviral drugs or recommended vaccines to treat or prevent FCoV infection. The plaque reduction neutralization test (PRNT) traditionally employed to assess immune responses and to screen therapeutic and vaccine candidates is time-consuming, low-throughput, and typically requires 2-3 days for the formation and manual counting of cytolytic plaques. Host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay. In addition, operator-to-operator variation can generate uncertainty in the results and digital records are not automatically created. To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. Host cell seeding density, microplate surface coating, virus concentration and incubation time, wash buffer and fluorescent labeling were optimized. Subsequently, this FCoV viral neutralization assay was used to explore immune correlates of protection using plasma from naturally FECV-infected cats. We demonstrate that the high-throughput viral neutralization assay using the Celigo Image Cytometer provides a robust and efficient method for the rapid screening of therapeutic antibodies, antiviral compounds, and vaccines. This method can be applied to various viral infectious diseases to accelerate vaccine and antiviral drug discovery and development.

Impact of oral probiotic Lactobacillus acidophilus vaccine strains on the immune response and gut microbiome of mice.

The potential role of probiotic bacteria as adjuvants in vaccine trials led to their use as nonparenteral live mucosal vaccine vectors. Yet, interactions between these vectors, the host and the microbiome are poorly understood. This study evaluates impact of three probiotic, Lactobacillus acidophilus, vector strains, and their interactions with the host's immune response, on the gut microbiome. One strain expressed the membrane proximal external region from HIV-1 (MPER). The other two expressed MPER and either secreted interleukin-1ß (IL-1ß) or expressed the surface flagellin subunit C (FliC) as adjuvants. We also used MPER with rice bran as prebiotic supplement. We observed a strain dependent, differential effect suggesting that MPER and IL-1ß induced a shift of the microbiome while FliC had minimal impact. Joint probiotic and prebiotic use resulted in a compound effect, highlighting a potential synbiotic approach to impact efficacy of vaccination. Careful consideration of constitutive adjuvants and use of prebiotics is needed depending on whether or not to target microbiome modulation to improve vaccine efficacy. No clear associations were observed between total or MPER-specific IgA and the microbiome suggesting a role for other immune mechanisms or a need to focus on IgA-bound, resident microbiota, most affected by an immune response.

Adjuvant Strategies for Lactic Acid Bacterial Mucosal Vaccines.

Lactic acid bacteria (LAB) are Gram-positive, acid-tolerant bacteria that have long been used in food fermentation and are generally recognized as safe (GRAS). LAB are a part of a normal microbiome and act as probiotics, improving the gastrointestinal microbiome and health when consumed. An increasing body of research has shown the importance of the microbiome on both mucosal immune heath and immune response to pathogens and oral vaccines. Currently, there are few approved mucosal vaccines, and most are attenuated viruses or bacteria, which necessitates cold chain, carries the risk of reversion to virulence, and can have limited efficacy in individuals with poor mucosal health. On account of these limitations, new types of mucosal vaccine vectors are necessary. There has been increasing interest and success in developing recombinant LAB as next generation mucosal vaccine vectors due to their natural acid and bile resistance, stability at room temperature, endogenous activation of innate and adaptive immune responses, and the development of molecular techniques that allow for manipulation of their genomes. To enhance the immunogenicity of these LAB vaccines, numerous adjuvant strategies have been successfully employed. Here, we review these adjuvant strategies and their mechanisms of action which include: Toll-like receptor ligands, secretion of bacterial toxins, secretion of cytokines, direct delivery to antigen presenting cells, and enterocyte targeting. The ability to increase the immune response to LAB vaccines gives them the potential to be powerful mucosal vaccine vectors against mucosal pathogens.

Effects of Regulatory T Cell Depletion on NK Cell Responses against Listeria monocytogenes in Feline Immunodeficiency Virus Infected Cats.

Regulatory T cells (Treg) are key players in the maintenance of peripheral tolerance, preventing autoimmune diseases and restraining chronic inflammatory diseases. Evidence suggests Treg cells and NK cells have important roles in feline immunodeficiency virus (FIV) pathogenesis; however, in vivo studies investigating the interplay between these two cell populations are lacking. We previously described innate immune defects in FIV-infected cats characterized by cytokine deficits and impaired natural killer cell (NK) and NK T cell (NKT) functions. In this study, we investigated whether in vivo Treg depletion by treatment with an anti-feline CD25 monoclonal antibody would improve the innate immune response against subcutaneous challenge with Listeria monocytogenes (Lm). Treg depletion resulted in an increased overall number of cells in Lm-draining lymph nodes and increased proliferation of NK and NKT cells in FIV-infected cats. Treg depletion did not normalize expression of perforin or granzyme A by NK and NKT cells, nor did Treg depletion result in improved clearance of Lm. Thus, despite the quantitative improvements in the NK and NKT cell responses to Lm, there was no functional improvement in the early control of Lm. CD1a+ dendritic cell percentages in the lymph nodes of FIV-infected cats were lower than in specific-pathogen-free control cats and failed to upregulate CD80 even when Treg were depleted. Taken together, Treg depletion failed to improve the innate immune response of FIV-infected cats against Lm and this may be due to dendritic cell dysfunction.

Mucosal Immune Response to Feline Enteric Coronavirus Infection.

Feline infectious peritonitis is a devastating, fatal disease of domestic cats caused by a pathogenic mutant virus derived from the ubiquitous feline enteric coronavirus (FECV). Infection by FECV is generally subclinical, and little is known about the mucosal immune response that controls and eliminates the virus. We investigated the mucosal immune response against FECV in an endemically infected breeding colony over a seven-month period. Thirty-three cats were grouped according to FECV seropositivity and fecal virus shedding into naïve/immunologically quiescent, convalescent and actively infected groups. Blood, fecal samples and colon biopsies were collected to assess the mucosal and systemic immunologic and virologic profile. Results showed that cats with active FECV infections have strong systemic IgG and mucosal IgA responses that wane after virus clearance. Significant FECV-specific mucosal T cell IFNγ responses were not detected in any of the three groups. A shift toward an inflammatory state in the mucosa was suggested by increased IL17:FoxP3 expression. However, no histologic abnormalities were observed, and no shifts in lymphocyte subpopulation phenotype or proliferation were noted. Together, the results suggest that control of FECV is mediated by humoral mucosal and systemic responses and that perturbations in the primary reservoir organ (colon) are minimal.

Lactobacillus Mucosal Vaccine Vectors: Immune Responses against Bacterial and Viral Antigens.

Lactic acid bacteria (LAB) have been utilized since the 1990s for therapeutic heterologous gene expression. The ability of LAB to elicit an immune response against expressed foreign antigens has led to their exploration as potential mucosal vaccine candidates. LAB vaccine vectors offer many attractive advantages: simple, noninvasive administration (usually oral or intranasal), the acceptance and stability of genetic modifications, relatively low cost, and the highest level of safety possible. Experimentation using LAB of the genus Lactobacillus has become popular in recent years due to their ability to elicit strong systemic and mucosal immune responses. This article reviews Lactobacillus vaccine constructs, including Lactobacillus species, antigen expression, model organisms, and in vivo immune responses, with a primary focus on viral and bacterial antigens.

Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform.

Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner. For selected bacterial isolates and mutants, we investigated the role of key innate immune pathways required for induction of innate and subsequent adaptive immune responses. Co-culture of murine macrophages with L. gasseri (strain NCK1785), L. acidophilus (strain NCFM), or NCFM-derived mutants-NCK2025 and NCK2031-elicited an M2b-like phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins. Through the use of macrophage genetic knockouts, we identified Toll-like receptor 2 (TLR2), the cytosolic nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, and the inflammasome-associated caspase-1 as contributors to macrophage activation, with NOD2 cooperating with caspase-1 to induce inflammasome derived interleukin (IL)-1ß in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine platform with surface expression of human immunodeficiency virus type 1 (HIV-1) Gag or membrane proximal external region (MPER), we demonstrated that NOD2 signaling is required for antigen-specific mucosal and systemic humoral responses. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a Lactobacillus acidophilus mucosal vaccine platform.

Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein.

Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.

Phenotypic and functional analysis of CD1a+ dendritic cells from cats chronically infected with feline immunodeficiency virus.

Numerous studies suggest dendritic cell (DC) dysfunction is central to the dysregulated immune response during HIV infection; however, in vivo studies are lacking. In the present study we used feline immunodeficiency virus (FIV) infection of cats as a model for HIV-1 infection to assess the maturation and function of dendritic cells, in vivo and in vitro. We compared CD1a+ DC migration, surface phenotype, endocytosis, mixed leukocyte reaction (MLR) and regulatory T cell (Treg) phenotype induction by CD1a+ cells isolated from lymph nodes of FIV-infected and control cats. Results showed that resident CD1a+ DC in lymph nodes of chronically FIV-infected cats are phenotypically mature, can stimulate normal primary T cell proliferation, override Treg suppression and do not skew toward Treg induction. In contrast, FIV infection had deleterious effects on antigen presentation and migratory capacity of CD1a+ cells in tissues.

A novel candidate vaccine for cytauxzoonosis inferred from comparative apicomplexan genomics.

Cytauxzoonosis is an emerging infectious disease of domestic cats (Felis catus) caused by the apicomplexan protozoan parasite Cytauxzoon felis. The growing epidemic, with its high morbidity and mortality points to the need for a protective vaccine against cytauxzoonosis. Unfortunately, the causative agent has yet to be cultured continuously in vitro, rendering traditional vaccine development approaches beyond reach. Here we report the use of comparative genomics to computationally and experimentally interpret the C. felis genome to identify a novel candidate vaccine antigen for cytauxzoonosis. As a starting point we sequenced, assembled, and annotated the C. felis genome and the proteins it encodes. Whole genome alignment revealed considerable conserved synteny with other apicomplexans. In particular, alignments with the bovine parasite Theileria parva revealed that a C. felis gene, cf76, is syntenic to p67 (the leading vaccine candidate for bovine theileriosis), despite a lack of significant sequence similarity. Recombinant subdomains of cf76 were challenged with survivor-cat antiserum and found to be highly seroreactive. Comparison of eleven geographically diverse samples from the south-central and southeastern USA demonstrated 91-100% amino acid sequence identity across cf76, including a high level of conservation in an immunogenic 226 amino acid (24 kDa) carboxyl terminal domain. Using in situ hybridization, transcription of cf76 was documented in the schizogenous stage of parasite replication, the life stage that is believed to be the most important for development of a protective immune response. Collectively, these data point to identification of the first potential vaccine candidate antigen for cytauxzoonosis. Further, our bioinformatic approach emphasizes the use of comparative genomics as an accelerated path to developing vaccines against experimentally intractable pathogens.

Infection with feline immunodeficiency virus alters intestinal epithelial transport and mucosal immune responses to probiotics.

HIV infection is associated with intestinal mucosal dysfunction and probiotics offer the therapeutic potential to enhance the mucosal barrier in HIV+ patients. To evaluate the response of immunocompromised hosts to probiotics, we orally administered Lactobacillus acidophilus to cats with chronic feline immunodeficiency virus (FIV) infection. FIV infection significantly affected transcellular, but not paracellular, transport of small molecules across the intestinal epithelium. Additionally, probiotic treatment of FIV+ cats resulted in changes in cytokine release and mucosal leukocyte percentages that were not paralleled in FIV- cats. These results suggest a novel role for FIV in upregulating transcellular transport across the gastrointestinal epithelial barrier and demonstrate the potential therapeutic use of probiotic bacteria to restore intestinal homeostasis.

TAK1 (MAP3K7) signaling regulates hematopoietic stem cells through TNF-dependent and -independent mechanisms.

A cytokine/stress signaling kinase Tak1 (Map3k7) deficiency is known to impair hematopoietic progenitor cells. However, the role of TAK1 signaling in the stem cell function of the hematopoietic system is not yet well defined. Here we characterized hematopoietic stem cells (HSCs) harboring deletion of Tak1 and its activators, Tak1 binding proteins 1 and 2 (Tab1 and Tab2) using a competitive transplantation assay in a mouse model. Tak1 single or Tab1/Tab2 double deletions completely eliminated the reconstitution activity of HSCs, whereas Tab1 or Tab2 single deletion did not cause any abnormality. Tak1 single or Tab1/Tab2 double deficient lineage-negative, Sca-1(+), c-Kit(+) (LSK) cells did not proliferate and underwent cell death. We found that Tnfr1 deficiency restored the reconstitution activity of Tak1 deficient bone marrow cells for 6-18 weeks. However, the reconstitution activity of Tak1- and Tnfr1-double deficient bone marrow cells declined over the long term, and the number of phenotypically identified long-term hematopoietic stem cells were diminished. Our results indicate that TAB1- or TAB2-dependent activation of TAK1 is required for maintenance of the hematopoietic system through two mechanisms: one is prevention of TNF-dependent cell death and the other is TNF-independent maintenance of long-term HSC.

Interleukin-15 activated bovine natural killer cells express CD69 and produce interferon-γ.

Interleukin (IL)-15 is an essential cytokine in natural killer (NK) cell development and survival. In humans, IL-15 shows overlapping properties with IL-2 due to partly shared receptors and signal transduction and both cytokines synergize equally well with IL-12 in the induction of interferon (IFN)-γ production from NK cells. Bovine NK cells however, have been reported to produce less IFN-γ after in vitro IL-12 stimulation when exposed to human IL-15 in comparison to bovine IL-2. We therefore wanted to determine if homologous IL-15 is needed for adequate stimulation of bovine NK cells. Biologically active recombinant bovine IL-15 (rbIL-15) produced in mammalian cells by the use of a modified expression vector stimulated NK cells to a dose-dependent IFN-γ production in the presence of IL-12. In contrast to earlier findings, we also detected potent IFN-γ production from bovine NK cells stimulated by human IL-15 and IL-12. Finally, we describe a monoclonal antibody recognizing bovine CD69 and show the expression of this early activation marker on bovine NK cells ex vivo and following rbIL-15 stimulation.

Cloning and establishment of canine desmocollin-1 as a major autoantigen in canine pemphigus foliaceus.

Pemphigus foliaceus (PF) is the most common antibody-mediated autoimmune skin disease of dogs. Desmoglein-1 (DSG1), the major human PF antigen, represents only a minor autoantigen in canine PF (cPF). A recent immunomapping study proposed desmocollin-1 (DSC1) as a relevant candidate autoantigen for cPF. To investigate this hypothesis, 85 cPF sera were screened for the presence of anti-DSC1 IgG using indirect immunofluorescence (IIF) on live canine DSC1-transfected 293T cells. Seventy-five sera contained detectable antikeratinocyte IgG on IIF using footpad substrate (IIFpos cPF), while 10 did not (IIFneg cPF). Sera from 35 healthy dogs, eight from exfoliative superficial pyoderma (ESP)-affected dogs and 21 dogs with non-PF autoimmune blistering skin diseases served as controls. All sera were tested concurrently by IIF on canine DSG1-transfected as well as nontransfected cells. None of the healthy dog or ESP sera labelled any of the transfected or nontransfected cells. Fifty-seven of 75 IIFpos cPF (86%) and 7/10 of IIFneg cPF sera (70%) contained detectable anti-DSC1 IgG. None of these sera recognized nontransfected cells. Five cPF sera (6%) recognized DSG1 in addition to DSC1. Finally, 5/21 (24%) sera from dogs with non-PF autoimmune blistering diseases contained low anti-DSC1 IgG titers. In 7/10 dogs (70%), from whom serial serum samples were collected during treatment, anti-DSC1 IgG titers decreased in parallel with the reduction in disease clinical severity. Altogether, these findings suggest that DSC1 is a major autoantigen in cPF.

In vivo assessment of natural killer cell responses during chronic feline immunodeficiency virus infection.

Accumulating evidence suggests that natural killer (NK) cells may have an important role in HIV-1 disease pathogenesis; however, in vivo studies are lacking. Feline immunodeficiency virus (FIV) infection of cats provides a valuable model to study NK cell function in vivo. The immune response against Listeria monocytogenes (Lm) is well characterized, allowing its use as an innate immune probe. We have previously shown that locally delivered IL-15 can improve Lm clearance in FIV-infected animals, and this correlated with an increase in NK cell number. In the present study, chronically FIV-infected and SPF-control cats were challenged with Lm by unilateral subcutaneous injection next to the footpad and then treated with 5-bromo-2'-deoxyuridine (BrdU). The Lm draining and contralateral control lymph nodes were evaluated for NK, NKT, CD4+ and CD8+ T cell number, proliferation, apoptosis, and NK cell function. Listeria monocytogenes burden was also assessed in both control and Lm draining lymph nodes. NK, NKT, CD4+ T and CD8+ T cells in the Lm-challenged lymph node of FIV-infected cats did not increase in number. In addition, after Lm challenge, NK cells from FIV-infected cats did not increase their proliferation rate, apoptosis was elevated, and perforin expression was not upregulated when compared to SPF-control cats. The failure of the NK cell response against Lm challenge in the draining lymph node of FIV-infected cats correlates with the delayed control and clearance of this opportunistic bacterial pathogen.