Ex vivo activation of the GCN2 pathway metabolically reprograms T cells, leading to enhanced adoptive cell therapy.

The manipulation of T cell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8+ T cells ex vivo using the general control non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment. The adoptive transfer of halo-treated CD8+ T cells into tumor-bearing mice led to robust tumor control and curative responses. Halo-treated T cells synergized in vivo with a 4-1BB agonistic antibody to control tumor growth in a mouse model resistant to immunotherapy. Importantly, treatment of human CD8+ T cells with halo resulted in similar metabolic and functional reprogramming. These findings demonstrate that activating the amino acid starvation response with the GCN2 agonist halo can enhance T cell metabolism and anti-tumor activity.

Tumor reactive γδ T cells contribute to a complete response to PD-1 blockade in a Merkel cell carcinoma patient.

Immunotherapies targeting PD-1/PD-L1 are now widely used in the clinic to treat a variety of malignancies. While most of the research on T cell exhaustion and PD-1 blockade has been focused on conventional αß T cells, the contribution of innate-like T cells such as γδ T cells to anti-PD-1/PD-L1 mediated therapy is limited. Here we show that tumor reactive γδ T cells respond to PD-1 blockade in a Merkel cell carcinoma (MCC) patient experiencing a complete response to therapy. We find clonally expanded γδ T cells in the blood and tumor after pembrolizumab treatment, and this Vγ2Vδ1 clonotype recognizes Merkel cancer cells in a TCR-dependent manner. Notably, the intra-tumoral γδ T cells in the MCC patient are characterized by higher expression of PD-1 and TIGIT, relative to conventional CD4 and CD8 T cells. Our results demonstrate that innate-like T cells could also contribute to an anti-tumor response after PD-1 blockade.

Coenzyme A fuels T cell anti-tumor immunity.

Metabolic programming is intricately linked to the anti-tumor properties of T cells. To study the metabolic pathways associated with increased anti-tumor T cell function, we utilized a metabolomics approach to characterize three different CD8+ T cell subsets with varying degrees of anti-tumor activity in murine models, of which IL-22-producing Tc22 cells displayed the most robust anti-tumor activity. Tc22s demonstrated upregulation of the pantothenate/coenzyme A (CoA) pathway and a requirement for oxidative phosphorylation (OXPHOS) for differentiation. Exogenous administration of CoA reprogrammed T cells to increase OXPHOS and adopt the CD8+ Tc22 phenotype independent of polarizing conditions via the transcription factors HIF-1α and the aryl hydrocarbon receptor (AhR). In murine tumor models, treatment of mice with the CoA precursor pantothenate enhanced the efficacy of anti-PDL1 antibody therapy. In patients with melanoma, pre-treatment plasma pantothenic acid levels were positively correlated with the response to anti-PD1 therapy. Collectively, our data demonstrate that pantothenate and its metabolite CoA drive T cell polarization, bioenergetics, and anti-tumor immunity.

The Roles of CD8+ T Cell Subsets in Antitumor Immunity.

Effector CD8+ T cells are typically thought to be a homogenous group of cytotoxic cells that produce interferon-(IFN) γ. However, recent findings have challenged this notion because multiple subsets of CD8+ T cells have been described, each with distinct effector functions and cytotoxic potential. These subsets, referred to as the Tc subsets, have also been detected in tumor microenvironments (TMEs), where they potentially influence the antitumor response and patient outcomes. In this review, we highlight the prevalence and roles of Tc subsets in the TME. We also discuss their therapeutic applications in the context of adoptive immunotherapy to treat cancer.

Cytotoxic CD4+ T Cells in Bladder Cancer-A New License to Kill.

CD4+ T cells with cytotoxic capability are increasingly recognized as potentially key actors in anti-tumor immunity. A new report in Cell elucidates the presence and potential role of a population of cytotoxic CD4+ T cells in bladder cancer using single cell sequencing technology.

Overproduction of IL-2 by Cbl-b deficient CD4+ T cells provides resistance against regulatory T cells.

Regulatory T cells are integral to the regulation of autoimmune and anti-tumor immune responses. However, several studies have suggested that changes in T cell signaling networks can result in T cells that are resistant to the suppressive effects of regulatory T cells. Here, we investigated the role of Cbl-b, an E3 ubiquitin ligase, in establishing resistance to Treg-mediated suppression. We found that the absence of Cbl-b, a negative regulator of multiple TCR signaling pathways, rendered T cells impartial to Treg suppression by regulating cytokine networks leading to improved anti-tumor immunity despite the presence of Treg cells in the tumor. Specifically, Cbl-b KO CD4+FoxP3- T cells hyper-produced IL-2 and together with IL-2 Rα upregulation served as an essential mechanism to escape suppression by Treg cells. Furthermore, we report that IL-2 serves as the central molecule required for cytokine-induced Treg resistance. Collectively our data emphasize the role of IL-2 as a key mechanism that renders CD4+ T cells resistant to the inhibitory effects of Treg cells.

IL6 Induces an IL22+ CD8+ T-cell Subset with Potent Antitumor Function.

CD8+ T cells can be polarized into several different subsets as defined by the cytokines they produce and the transcription factors that govern their differentiation. Here, we identified the polarizing conditions to induce an IL22-producing CD8+ Tc22 subset, which is dependent on IL6 and the aryl hydrocarbon receptor transcription factor. Further characterization showed that this subset was highly cytolytic and expressed a distinct cytokine profile and transcriptome relative to other subsets. In addition, polarized Tc22 were able to control tumor growth as well as, if not better than, the traditional IFNγ-producing Tc1 subset. Tc22s were also found to infiltrate the tumors of human patients with ovarian cancer, comprising up to approximately 30% of expanded CD8+ tumor-infiltrating lymphocytes (TIL). Importantly, IL22 production in these CD8+ TILs correlated with improved recurrence-free survival. Given the antitumor properties of Tc22 cells, it may be prudent to polarize T cells to the Tc22 lineage when using chimeric antigen receptor (CAR)-T or T-cell receptor (TCR) transduction-based immunotherapies.

Activation of Peroxisome Proliferator-Activated Receptors α and δ Synergizes with Inflammatory Signals to Enhance Adoptive Cell Therapy.

Memory CD8+ T cells (Tmem) are superior mediators of adoptive cell therapy (ACT) compared with effector CD8+ T cells (Teff) due to increased persistence in vivo. Underpinning Tmem survival is a shift in cellular metabolism away from aerobic glycolysis towards fatty acid oxidation (FAO). Here we investigated the impact of the peroxisome proliferator-activated receptor (PPAR) agonist GW501516 (GW), an agent known to boost FAO in other tissues, on CD8+ T-cell metabolism, function, and efficacy in a murine ACT model. Via activation of both PPARα and PPARδ/ß, GW treatment increased expression of carnitine palmitoyl transferase 1a, the rate-limiting enzyme of FAO, in activated CD8+ T cells. Using a metabolomics approach, we demonstrated that GW increased the abundance of multiple different acylcarnitines, consistent with enhanced FAO. T cells activated in the presence of GW and inflammatory signals, either mature dendritic cells or IL12, also demonstrated enhanced production of IFNγ and expression of T-bet. Despite high expression of T-bet, a characteristic of short-lived effector cells, GW-treated cells demonstrated enhanced persistence in vivo and superior efficacy in a model of ACT. Collectively, these data identify combined PPARα and PPARδ/ß agonists as attractive candidates for further studies and rapid translation into clinical trials of ACT. SIGNIFICANCE: Dual activation of peroxisome proliferator-activated receptors α and δ improves the efficacy of adoptive cell therapy by reprogramming T-cell metabolism and cytokine expression.

Evaluation of a polysaccharide conjugate vaccine to reduce colonization by Campylobacter jejuni in broiler chickens.

BACKGROUND: Campylobacter jejuni is a leading bacterial cause of food-borne illness in humans. Symptoms range from mild gastroenteritis to dysentery. Contaminated chicken meat is the most common cause of infection. Broiler chickens become colonized with high numbers of C. jejuni in the intestinal tract, but do not become clinically ill. Vaccination of broiler chicks to control colonization by C. jejuni is challenging because immune function is limited in the first 2 weeks post-hatch and immune suppressive maternal antibodies are common. In addition, there is little time for induction of immunity, since broilers reach slaughter weight by 5-6 weeks of age. In the current study the immunogenicity of a C. jejuni capsular polysaccharide-diphtheria toxoid conjugated vaccine (CPSconj), administered subcutaneously with various adjuvants was assessed and the efficacy of vaccination for reducing cecal colonization after experimental challenge was evaluated by determining colony-forming units (CFU) of C. jejuni in cecal contents. RESULTS: The CPSconj vaccine was immunogenic when administered as three doses at 3, 4 and 5 weeks of age to specific pathogen free chicks lacking maternal antibodies (seroconversion rates up to 75%). Commercial broiler chicks (having maternal antibodies) receiving two doses of CPSconj vaccine at 7 and 21 days of age did not seroconvert before oral challenge at 29 days, but 33% seroconverted post challenge; none of the placebo-injected, challenged birds seroconverted. Vaccinated birds had significantly lower numbers of C. jejuni in cecal contents than control birds at necropsy (38 days of age). CFU of C. jejuni did not differ significantly among groups of birds receiving CPSconj vaccine with different adjuvants. In two trials, the mean reduction in CFU associated with vaccination was 0.64 log10 units. CONCLUSIONS: The CPSconj vaccine was immunogenic in chicks lacking maternal antibodies, vaccinated beginning at 3 weeks of age. In commercial broiler birds (possessing maternal antibodies) vaccinated at 7 and 21 days of age, 33% of birds seroconverted by 9 days after challenge, and there was a modest, but significant, reduction in cecal counts of C. jejuni. Further studies are needed to optimize adjuvant, route of delivery and scheduling of administration of this vaccine.

Deficiency of MALT1 paracaspase activity results in unbalanced regulatory and effector T and B cell responses leading to multiorgan inflammation.

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

Expression profiles of antiviral response genes in chicken bursal cells stimulated with Toll-like receptor ligands.

Cells of the adaptive immune system express Toll-like receptors (TLRs) and are able to respond to TLR ligands. With this in mind, the goal of the current study was to determine the expression of antiviral response genes in the cells of the chicken bursa of Fabricius (BF) to stimulation with TLR ligands. We investigated initially the response of bursal B cells to CpG-ODN, lipopolysaccharide (LPS) and poly(I:C) treatment. The expression level of type I interferons (IFNs) and interferon regulatory factor 7 (IRF7) did not differ between CpG-ODN and LPS treated groups compared to the non-stimulated cells. Poly(I:C) was the only TLR ligand, which has induced significant expression of antiviral innate immune response genes from bursal cells. Further in vitro and in vivo studies need to examine the efficacy of these antiviral responses against avian viruses.

Effects of ligands for Toll-like receptors 3, 4, and 21 as adjuvants on the immunogenicity of an avian influenza vaccine in chickens.

Avian influenza viruses (AIV) are of great concern to the worldwide community as well as the poultry industry. Although existing vaccines are successful in limiting the spread of the virus, these vaccines do not eliminate virus shedding into the environment. As a result, it is of great importance to enhance the efficacy of existing AIV vaccines. Therefore, the objective of the present study was to utilize the immunostimulatory Toll-like receptor ligands poly I:C, lipopolysaccharide (LPS), and CpG DNA motifs, either alone or in combination with each other, as adjuvants to enhance the immunogenicity of an inactivated AIV vaccine. Chickens were vaccinated twice, 14 days apart. Antibody-mediated responses were assessed by collected sera and lacrimal secretions, while cell-mediated immunity was assessed by stimulating splenocytes from vaccinated chickens in vitro with the vaccine antigen. The results suggest that CpG alone served as the best single-ligand adjuvant compared to poly I:C or LPS, as it significantly enhanced antibody-mediated responses, as determined by enzyme-linked immunosorbant assay. Furthermore, upon combining CpG with poly I:C, a robust antibody-mediated and cell-mediated immune response was elicited, resulting in an enhanced hemagglutination inhibition titer and splenocyte proliferation respectively. Future studies may be aimed at assessing the efficacy of the poly I:C and CpG combination adjuvant in protecting against AIV infection.

Avian influenza virus vaccines containing Toll-like receptors 2 and 5 ligand adjuvants promote protective immune responses in chickens.

Vaccination remains a useful means for the control of avian influenza viruses (AIV) in chickens. Current vaccines can protect chickens from morbidity and mortality. However, they do not eliminate virus shedding into the environment. Therefore, novel measures must be considered in order to enhance the immunogenicity of AIV vaccines, such as through the administration of immunostimulatory compounds. One such group of compounds is Toll-like receptor (TLR) ligands, such as bacterial flagellin, as well as synthetic lipopeptides such as Pam3CSK4. The objective of the present study was to assess the adjuvant potential of TLR2 and TLR5 ligands flagellin and Pam3 respectively. Chickens were vaccinated twice with an inactivated H4N6 AIV vaccine, 14 days apart. Antibody-mediated responses were assessed in sera and lacrimal secretions, while cell-mediated immune response was assessed by stimulating splenocytes from vaccinated chickens in vitro with the vaccine antigen. To evaluate vaccine efficacy, chickens were challenged with the H4N6 virus, and virus shedding was assessed on day 7 post-challenge. The results suggest that both ligands significantly enhanced antigen-specific IgY antibodies, while only the Pam3 adjuvant induced greater IgM and IgA antibody levels. Chickens receiving the flagellin adjuvant had significantly higher IgY responses, as well as significantly higher hemagglutination-inhibition antibody titers compared to the no adjuvant control. With respect to cell-mediated responses, splenocytes isolated from chickens that received either TLR ligand adjuvant proliferated in response to an in vitro stimulation with vaccine antigens. Lastly, chickens receiving vaccines containing either flagellin or Pam3 adjuvants were partially protected from an experimental AIV challenge and shed significantly less virus compared to controls. Future studies may be aimed at examining the efficacy of Pam3 and flagellin adjuvants for highly pathogenic AIV strains.

Toso controls encephalitogenic immune responses by dendritic cells and regulatory T cells.

The ability to mount a strong immune response against pathogens is crucial for mammalian survival. However, excessive and uncontrolled immune reactions can lead to autoimmunity. Unraveling how the reactive versus tolerogenic state is controlled might point toward novel therapeutic strategies to treat autoimmune diseases. The surface receptor Toso/Faim3 has been linked to apoptosis, IgM binding, and innate immune responses. In this study, we used Toso-deficient mice to investigate the importance of Toso in tolerance and autoimmunity. We found that Toso(-/-) mice do not develop severe experimental autoimmune encephalomyelitis (EAE), a mouse model for the human disease multiple sclerosis. Toso(-/-) dendritic cells were less sensitive to Toll-like receptor stimulation and induced significantly lower levels of disease-associated inflammatory T-cell responses. Consistent with this observation, the transfer of Toso(-/-) dendritic cells did not induce autoimmune diabetes, indicating their tolerogenic potential. In Toso(-/-) mice subjected to EAE induction, we found increased numbers of regulatory T cells and decreased encephalitogenic cellular infiltrates in the brain. Finally, inhibition of Toso activity in vivo at either an early or late stage of EAE induction prevented further disease progression. Taken together, our data identify Toso as a unique regulator of inflammatory autoimmune responses and an attractive target for therapeutic intervention.

Characterization of responses initiated by different Toll-like receptor 2 ligands in chicken spleen cells.

Toll-like receptors (TLRs) are pattern recognition receptors that mediate host responses to pathogens by promoting cellular activation and the production of cytokines. Ligands for TLRs are conserved structural motifs of pathogens termed pathogen-associated molecular patterns. In the case of TLR2, these ligands include peptidoglycan, lipomannan and lipopeptides. In mammals, it has been shown that different TLR2 ligands induce distinct cytokine responses. However, whether a similar phenomenon occurs in chickens remains to be determined. To this end, chicken splenocytes were stimulated with three different TLR2 ligands: Pam3CSK4, FSL-1 and lipomannan, and the relative gene expression of several cytokines was quantified at 2, 6 and 18h post-stimulation. The results suggest that Pam3 and FSL-1 modulate the kinetics of the pro-inflammatory cytokine response differently, as Pam3 induced a robust interleukin (IL)-1ß response, while FSL-1 induced an early and prolonged up-regulation of IL-8. Furthermore, it appears that all three TLR2 ligands induce a mixed T-helper (TH) 1 and 2-like response, as characterized by the up-regulation of IFN-γ, IL-12, IL-4 and IL-13. In conclusion, we have demonstrated that different TLR2 ligands may induce different cytokine responses in chicken splenocytes. Future studies may be aimed at examining the immunomodulating effects of these ligands in vivo.

Small interfering RNA-mediated knockdown of chicken interferon-γ expression.

Interferon (IFN)-γ is a cytokine with a variety of functions, including direct antiviral activities and the capacity to polarize T-cells. However, there is limited information available about the function of this cytokine in the avian immune system. To gain a better understanding of the biological relevance of IFN-γ in chicken immunity, gain-of-function (upregulation) and loss-of-function (downregulation) studies need to be conducted. RNA interference (RNAi), a technique employed for downregulating gene expression, is mediated by small interfering RNA (siRNA), which can trigger sequence-specific gene silencing. In this regard, sequence specificity and delivery of siRNA molecules remain critical issues, especially to cells of the immune system. Various direct and indirect approaches have been employed to deliver siRNA, including the use of viral vectors. The objectives of the present study were to determine whether RNAi could effectively downregulate expression of chicken IFN-γ in vitro, and investigate the feasibility of recombinant adeno-associated virus to deliver siRNA in vitro as well. Three 27-mer Dicer substrate RNAs were selected based on the chicken IFN-γ coding sequence and transfected into cells or delivered using a recombinant avian adeno-associated virus (rAAAV) into a chicken fibroblast cell line expressing chIFN-γ. The expression of chIFN-γ transcripts was significantly downregulated when a cocktail containing all three siRNAs was used. Expression of endogenous IFN-γ was also significantly downregulated in primary cells after stimulation with a peptide. Further, significant suppression of IFN-γ transcript was also observed in vitro in cells that were treated with rAAAV, expressing siRNA targeting IFN-γ. Off-target effects in the form of triggering IFN responses by RNAi, including expression of chicken 2',5'-oligoadenylate synthetase and IFN-α, were also examined. Our results suggest that siRNAs selected were effective at downregulating IFN-γ in vitro both when delivered directly as well as when expressed by an rAAAV-based vector.

Induction of chicken cytokine responses in vivo and in vitro by lipooligosaccharide of Campylobacter jejuni HS:10.

Campylobacter jejuni is a pathogen of the gastrointestinal tract of humans, but colonizes chickens for prolonged periods without causing disease. It is unclear what host and bacterial mechanisms maintain a non-inflammatory state in chickens. The present work was undertaken to characterize cytokine responses of chickens to purified lipooligosaccharide (LOS) of C. jejuni HS:10. Chickens were injected with purified LOS, and expression of interleukin (IL)-1ß (pro-inflammatory cytokine), IL-8 (pro-inflammatory chemokine), interferon (IFN)γ (Th1-like cytokine), IL-10 (immune regulatory/anti-inflammatory cytokine) and IL-13 (Th2-like cytokine) was evaluated in spleen using quantitative RT-PCR, up to 24h post-injection. In an in vitro study, splenocytes were incubated with LOS, and cytokine expression followed up to 18 h. Chickens injected with LOS had increased expression of IL-1ß up to 24h later. Expression of IL-8 was significantly increased at 2h but then declined below baseline. Expression of IFNγ and IL-10 was increased significantly at 2h, but declined thereafter. Splenocytes incubated with LOS had increased expression of IL-1ß and IL-8 up to 18 h of incubation. Expression of IFNγ was increased at 6 and 18 h, IL-10 was increased at 2h, but expression of IL-13 did not differ significantly up to 18h. It is concluded that LOS of C. jejuni can induce expression of pro-inflammatory IL-1ß and IL-8, as well as IFNγ and IL-10 in chickens. More extensive studies with more prolonged exposure to LOS are needed to further clarify the interaction between C. jejuni and the chicken host.

Chicken erythrocytes respond to Toll-like receptor ligands by up-regulating cytokine transcripts.

Avian erythrocytes are nucleated cells of myeloid origin that are able to actively transcribe and translate proteins. Although their role in gas exchange and transportation has been well described, it has recently been shown that chicken erythrocytes produce cytokines in response to Toll-like receptor (TLR) ligands, which raises the possibility that they also contribute to host immunity. To this end, the objective of the study was to gain some further insight into the immunological role of erythrocytes by identifying the repertoire of TLRs that they express and to elucidate their responses to the TLR3 and TLR21 ligands poly I:C and CpG oligodeoxynucleotide (ODN), respectively. The results suggest that erythrocytes constitutively express transcripts for TLRs 2, 3, 4, 5, and 21, as well as for many immunological genes including type I interferons (IFN) and interleukin (IL)-8. Moreover, it was found that treatment with both poly I:C and CpG ODN up-regulated transcripts for type I IFNs, while only poly I:C up-regulated IL-8 transcripts and enhanced the production of nitrite. Future studies may be aimed at further characterizing the immunological role of erythrocytes.

Immunostimulatory properties of Toll-like receptor ligands in chickens.

Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that have been identified in mammals and avian species. Ligands for TLRs are typically conserved structural motifs of microorganisms termed pathogen-associated molecular patterns (PAMPs). Several TLRs have been detected in many cell subsets, such as in macrophages, heterophils and B cells, where they mediate host-responses to pathogens by promoting cellular activation and the production of cytokines. Importantly, TLR ligands help prime a robust adaptive immune response by promoting the maturation of professional antigen presenting cells. These properties make TLR ligands an attractive approach to enhance host-immunity to pathogens by administering them either prophylactically or in the context of a vaccine adjuvant. In this review, we discuss what is known about the immunostimulatory properties of TLR ligands in chickens, both at the cellular level as well as in vivo. Furthermore, we highlight previous successes in exploiting TLR ligands to protect against several pathogens including avian influenza virus, Salmonella, Escherichia coli, and Newcastle disease Virus.

Response of older laying hens to an Escherichia coli lipopolysaccharide challenge when fed diets with or without supplemental folic acid.

Folic acid plays a key role in nucleic acids and protein synthesis, and has been associated with anti-inflammatory effects in LPS-induced infections. To this end, a study was conducted to investigate the effects of dietary folic acid (FA) supplementation in older laying hens (58 to 66 wk of age) challenged with Escherichia coli lipopolysaccharide (LPS). A total of 24 Shaver White laying hens at 58 wk were fed 2 diets. The diets were wheat-soybean-based, with either 0 or 4 mg of supplemental FA per kg of diet. After 8 wk of feeding and at 66 wk, the hens were injected intravenously with 8 mg of LPS or saline per kg of BW. Four hours after injection, blood was collected and hens were euthanized to obtain spleen and cecal tonsils. The T cell subsets in the blood and the spleen (CD4+ and CD8+), total IgG, and biochemical constituents (total protein, albumin, globulin, and fibrinogen) were not influenced (P > 0.05) by dietary FA supplementation. However, LPS injection decreased (P < 0.05) biochemical constituents, CD4+, and CD8+ cells in the blood, whereas CD4+:CD8+ ratio and total IgG increased (P < 0.05), and fibrinogen was not influenced. Gene expression in the spleen and cecal tonsils was not influenced by dietary FA supplementation except a diet × challenge interaction for interleukin (IL)-8 in the spleen; IL-8 decreased in FA-fed hens that were treated with LPS. Also, FA supplementation decreased the expression of IL-8 in cecal tonsils. Relative to saline-injected hens, expression of IL-1ß, interferon-γ, and IL-10 increased in the LPS-injected hens in the spleen and cecal tonsils, IL-8 increased in LPS-injected hens only in the cecal tonsils, whereas Toll-like receptor 4, IL-4, IL-17, and IL-18 increased in the LPS-injected hens only in the spleen; however, LPS decreased expression of IL-13 in the cecal tonsils. In conclusion, FA did not affect inflammatory responses in older laying hens; more studies are required to investigate possible protective effects of FA in laying hens.