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Papers by Sara Hamilton

Research paper thumbnail of Lung ILC2 responses to <i>Alternaria alternata</i> fungal allergen are blunted in “dirty” mice with physiologically transmitted murine microbes

Journal of Immunology, May 1, 2020

The objective of this study was to test the hypothesis that the increasing global incidence of al... more The objective of this study was to test the hypothesis that the increasing global incidence of allergy and atopy are due in part to improved hygiene and decreased microbial exposure. We have adopted a novel mouse model of normalized microbial exposure to test the impact of immune experience on subsequent responses to airway allergens. In this model, specific pathogen free (SPF) B6 mice are cohoused with mice from pet stores and become “dirty” – many commensals and pathogens are transmitted through cohousing and influence the immune cell populations systemically and in the lungs. We treated mice intranasally with a single dose of A. alternata fungal extract (Alt) and assessed production of type 2 cytokines. IL5 and IL13 levels in the lungs and bronchoalveolar lavage fluid were dramatically elevated by Alt in SPF mice but were not significantly increased by Alt in dirty mice. Type 2 innate lymphoid cells (ILC2) are the cells responsible for IL5 and IL13 after acute Alt treatment, and interestingly the number of lung ILC2 was unaltered by cohousing and their activation status appeared similar in both housing conditions. We treated mice with recombinant IL33, the alarmin released by lung epithelial cells in response to allergens, and the results suggest an impaired response to IL33 by dirty lung ILC2. In a repeated Alt exposure model, ILC2 cells expanded in SPF lungs and recruited eosinophils, neutrophils, and T cells. Most dirty mouse lungs contained these cell populations at steady state but were only modestly recruited with repeated Alt exposure. Lung function experiments are in progress. This study suggests that increased microbial exposure leads to more type-2 associated immune cells in the lungs, however responses to airway allergens are dampened.

Research paper thumbnail of Rapid cell cycling of both circulating and resident memory CD8+ T cells driven by common gamma chain-dependent cytokines

Journal of Immunology, May 1, 2021

Diverse populations of resident leukocytes live and self-renew within the tissues of the body, bu... more Diverse populations of resident leukocytes live and self-renew within the tissues of the body, but the mechanisms controlling their proliferation are poorly understood. Among these populations are many memory CD8+ T cells, which are found throughout the body and can locally control reinfection. A poor ability of vaccines to elicit stable and appropriate tissue-resident CD8+ T cell populations has likely contributed to long-standing challenges in vaccinating against several important pathogens. The factors required to support TRM proliferation in homeostasis are not well understood. We propose that proliferating TRMs flexibly use the available members of the common gamma chain-(γC)-dependent cytokine family to persist as bystanders during other immune responses. We have found that many γC-dependent cytokines can mediate cell-intrinsic signaling to drive proliferation of both circulating and resident memory CD8+ T cells of known antigen specificities without cognate antigen. Via transcriptional analysis of resting and in vivo cytokine-stimulated TRMs, we are elucidating downstream signaling and the gene expression signature of cytokine-driven TRM proliferation. We have also found that memory CD8+ T cells proliferate similarly in response to exogenous cytokines or unrelated pathogens, consistent with pathogen-elicited γC-dependent cytokine signals driving memory CD8+ T cell proliferation. These data support the concept that TRMs use γC-dependent cytokines produced during infections to support enhanced proliferation, likely to promote their maintenance. Our findings may lead to novel methods to therapeutically enhance TRM immunity.

Research paper thumbnail of IL-15 complex-stimulated NK cells protect mice from cerebral malaria

Journal of Immunology, May 1, 2016

Cerebral malaria (CM) is one of the most lethal complications of Plasmodium falciparum infection,... more Cerebral malaria (CM) is one of the most lethal complications of Plasmodium falciparum infection, responsible for a large fraction of the nearly one million malaria-related deaths annually. Infection of susceptible mouse strains such as C57BL/6 with Plasmodium berghei ANKA (PbA) induces a fatal neurological syndrome from 6–10 days post-infection (dpi) resulting from CD8 T cell-mediated damage to the CNS. We found that treatment of C57BL/6 mice with interleukin (IL)-15 complexes (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) prevented the development of PbA-induced CM. Rescue from CM was not associated with reduced parasitemia at 5 dpi. Instead, IL-15C treatment resulted in reduced CD8 T cell activation in the brain at 6 dpi and reduced blood brain barrier breakdown. In addition, adoptive transfer of IL-15C-stimulated natural killer (NK) cells (but not CD8+ T cells) was sufficient to prevent CM. IL-15C and similar complexes formed with IL-2 (IL-2C; IL-2 bound to the anti-IL-2 S4B6 antibody) both cause robust expansion and activation of NK cells but, unexpectedly, NK cells from mice treated with IL-2C failed to protect against CM. Comparative RNAseq analysis of IL-15C and IL-2C-treated NK cells identified novel gene expression patterns, demonstrating previously unappreciated differences between these cytokine complex signaling cascades in NK cells; the functional significance of these differences is being assessed. These data indicate that NK cells – which are typically involved in promoting inflammatory responses – can restrain damaging immune responses, and that differences between the activation of NK cells by IL-2C and IL-15C regulates their capacity to control the inflammatory response to blood stage malaria infection.

Research paper thumbnail of IL-4 sensitivity shapes the peripheral CD8+ T cell pool and response to infection

Journal of Immunology, May 1, 2016

Research paper thumbnail of Responsiveness to interleukin-15 therapy is shared between tissue-resident and circulating memory CD8 + T cell subsets

Proceedings of the National Academy of Sciences

Interleukin-15 (IL-15) is often considered a central regulator of memory CD8 + T cells, based pri... more Interleukin-15 (IL-15) is often considered a central regulator of memory CD8 + T cells, based primarily on studies of recirculating subsets. However, recent work identified IL-15–independent CD8 + T cell memory populations, including tissue-resident memory CD8 + T cells (T RM ) in some nonlymphoid tissues (NLTs). Whether this reflects the existence of IL-15–insensitive memory CD8 + T cells is unclear. We report that IL-15 complexes (IL-15c) stimulate rapid proliferation and expansion of both tissue-resident and circulating memory CD8 + T cell subsets across lymphoid and nonlymphoid tissues with varying magnitude by tissue and memory subset, in some sites correlating with differing levels of the IL-2Rβ. This was conserved for memory CD8 + T cells recognizing distinct antigens and elicited by different pathogens. Following IL-15c–induced expansion, divided cells contracted to baseline numbers and only slowly returned to basal proliferation, suggesting a mechanism to transiently amplif...

Research paper thumbnail of Defining the role of CD69 in the formation of resident memory CD8+ T cells

Journal of Immunology, 2018

Resident memory CD8+ T cells (T) reside in nonlymphoid tissues. There, they play a key role in pr... more Resident memory CD8+ T cells (T) reside in nonlymphoid tissues. There, they play a key role in preventing reinfection by exerting cytotoxic and inflammatory functions upon exposure to previously encountered pathogens. CD69 is often used as a definitive marker of T cells. CD69's interaction with the G-proteincoupled receptor S1PR1 has been identified as one mechanism by which CD69 can regulate tissue residency. However, the functional requirement for CD69 in promoting the generation and maintenance of CD8+ T under a wide variety of circumstances remains unclear. We explored the role of CD69 in tissue residency using co-transfer of antigen specific CD69 sufficient and deficient CD8+ T cells in the context of acute LCMV, Influenza, and VSV infections. Strikingly, we found that CD69 was not necessary for T establishment in most tissues, although it can promote T localization under some circumstances. This seems to be influenced by the focal point of infection. Interestingly, the kidney appears to rely on CD69 for tissue residency with every model pathogen examined. We propose that the requirement for CD69 is context dependent rather than absolute, and that a combination of factors, including tissue microenvironment and infectious agent, dictate CD69's influence on development of CD8+ resident memory.

Research paper thumbnail of Contributions of natural killer cells to the immune response against Plasmodium

Malaria Journal, 2019

Natural killer (NK) cells are important innate effector cells that are well described in their ab... more Natural killer (NK) cells are important innate effector cells that are well described in their ability to kill virally-infected cells and tumors. However, there is increasing appreciation for the role of NK cells in the control of other pathogens, including intracellular parasites such asPlasmodium, the cause of malaria. NK cells may be beneficial during the early phase ofPlasmodiuminfection—prior to the activation and expansion of antigen-specific T cells—through cooperation with myeloid cells to produce inflammatory cytokines like IFNγ. Recent work has defined howPlasmodiumcan activate NK cells to respond with natural cytotoxicity, and inhibit the growth of parasites via antibody-dependent cellular cytotoxicity mechanisms (ADCC). A specialized subset of adaptive NK cells that are negative for the Fc receptor γ chain have enhanced ADCC function and correlate with protection from malaria. Additionally, production of the regulatory cytokine IL-10 by NK cells prevents overt pathology ...

Research paper thumbnail of The Functional Requirement for CD69 in Establishment of Resident Memory CD8+ T Cells Varies with Tissue Location

The Journal of Immunology, 2019

Recent studies have characterized populations of memory CD8+ T cells that do not recirculate thro... more Recent studies have characterized populations of memory CD8+ T cells that do not recirculate through the blood but are, instead, retained in nonlymphoid tissues. Such CD8+ tissue resident memory T cells (TRM) are critical for pathogen control at barrier sites. Identifying TRM and defining the basis for their tissue residency is therefore of considerable importance for understanding protective immunity and improved vaccine design. Expression of the molecule CD69 is widely used as a definitive marker for TRM, yet it is unclear whether CD69 is universally required for producing or retaining TRM. Using multiple mouse models of acute immunization, we found that the functional requirement for CD69 was highly variable, depending on the tissue examined, playing no detectable role in generation of TRM at some sites (such as the small intestine), whereas CD69 was critical for establishing resident cells in the kidney. Likewise, forced expression of CD69 (but not expression of a CD69 mutant un...

Research paper thumbnail of Targeting glutamine metabolism rescues mice from late-stage cerebral malaria

Proceedings of the National Academy of Sciences of the United States of America, Jan 5, 2015

The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a c... more The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15-25% in African children despite effective antimalarial chemotherapy. There are no adjunctive treatments for CM, so there is an urgent need to identify new targets for therapy. Here we show that the glutamine analog 6-diazo-5-oxo-l-norleucine (DON) rescues mice from CM when administered late in the infection a time at which mice already are suffering blood-brain barrier dysfunction, brain swelling, and hemorrhaging accompanied by accumulation of parasite-specific CD8(+) effector T cells and infected red blood cells in the brain. Remarkably, within hours of DON treatment mice showed blood-brain barrier integrity, reduced brain swelling, decreased function of activated effector CD8(+) T cells in the brain, and levels of brain metabolites that resembled those in uninfected mice. These results suggest DON as a strong candidate for an effective adjunctive therapy for CM...

Research paper thumbnail of Neutrophil Involvement in Cross-Priming CD8+ T Cell Responses to Bacterial Antigens

The Journal of Immunology, 2004

Substantial CD8+ T cell responses are generated after infection of mice with recombinant Listeria... more Substantial CD8+ T cell responses are generated after infection of mice with recombinant Listeria monocytogenes strains expressing a model epitope (lymphocytic choriomeningitis virus NP118–126) in secreted and nonsecreted forms. L. monocytogenes gains access to the cytosol of infected cells, where secreted Ags can be accessed by the endogenous MHC class I presentation pathway. However, the route of presentation of the nonsecreted Ag in vivo remains undefined. In this study we show that neutrophil-enriched peritoneal exudate cells from L. monocytogenes-infected mice can serve as substrates for in vitro cross-presentation of both nonsecreted and secreted Ag by dendritic cells as well as for in vivo cross-priming of CD8+ T cells. In addition, specific neutrophil depletion in vivo by low dose treatment with either of two Ly6G-specific mAb substantially decreased the relative CD8+ T cell response against the nonsecreted, but not the secreted, Ag compared with control Ab-treated mice. Thu...

Research paper thumbnail of Quantitation of CD8+ T Cell Expansion, Memory, and Protective Immunity After Immunization with Peptide-Coated Dendritic Cells

The Journal of Immunology, 2002

Dendritic cells (DCs) are potent APCs for naive CD8+ T cells and are being investigated as vaccin... more Dendritic cells (DCs) are potent APCs for naive CD8+ T cells and are being investigated as vaccine delivery vehicles. In this study, we examine the CD8+ T cell response to defined peptides from Listeria monocytogenes (LM), lymphocytic choriomeningitis virus, and murine CMV coated singly and in combination onto mature bone marrow-derived DCs (BMDCs). We show that immunization of mice with 2 × 105 mature BMDCs coated with multiple MHC class I peptides generates a significant Ag-specific CD8+ T cell response in both the spleen and nonlymphoid organs. This immunization resulted in a peptide-specific hierarchy in the magnitude of CD8+ T cell priming and noncoordinate kinetics in response to different peptide epitopes. Kinetics were not exclusively due to specific characteristics of the MHC class I molecule, and were not altered in an Ag-independent manner by concurrent LM infection. Mice immunized with listeriolysin O 91–99-coated BMDCs are protected against high dose challenge with viru...

Research paper thumbnail of Listeria monocytogenesInfection Overcomes the Requirement for CD40 Ligand in Exogenous Antigen Presentation to CD8+ T Cells

The Journal of Immunology, 2001

In vivo priming of CD8+ T lymphocytes against exogenously processed model Ags requires CD4+ T cel... more In vivo priming of CD8+ T lymphocytes against exogenously processed model Ags requires CD4+ T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4+ T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8+ T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8+ T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8+ T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8+ T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immuniz...

Research paper thumbnail of Impaired Assembly yet Normal Trafficking of MHC Class I Molecules in Tapasin Mutant Mice

Immunity, 2000

sis, nascent class I heavy chains (HCs) associate with calnexin, an ER resident chaperone. The cl... more sis, nascent class I heavy chains (HCs) associate with calnexin, an ER resident chaperone. The class I HCs then bind with ␤ 2-microglobulin (␤ 2 m), and HC-␤ 2 m are

Research paper thumbnail of CD8+-T-Cell Response to Secreted and Nonsecreted Antigens Delivered by RecombinantListeria monocytogenesduring Secondary Infection

Infection and Immunity, 2002

ABSTRACTUnderstanding how existing antivector immunity impacts live vaccine delivery systems is c... more ABSTRACTUnderstanding how existing antivector immunity impacts live vaccine delivery systems is critical when the same vector system may be used to deliver different antigens. We addressed the impact of antivector immunity, elicited by immunization with attenuatedactA-deficientListeria monocytogenes, on the CD8+-T-cell response to a well-characterized lymphocytic choriomeningitis virus epitope, NP118-126, delivered by infection with recombinantL. monocytogenes. Challenges of immune mice withactA-deficient and with wild-type recombinantL. monocytogenesgenerated similar numbers of CD8+T cells specific for the NP118-126 epitope. High-dose immunization withactA-deficientL. monocytogenesresulted in substantial numbers of CD8+T cells specific for theL. monocytogenesLLO91-99 epitope in the effector and memory stages of the T-cell response. Challenge of these immune mice with recombinantL. monocytogenesresulted in rapid control of the infection and decreased CD8+-T-cell responses against bo...

Research paper thumbnail of Environmental conservation: bystander CD4 T cells keep CD8 memories fresh

Nature Immunology, Sep 1, 2004

CD4+ T cells reportedly program CD8+ T cells to develop into suitable memory cells during the pri... more CD4+ T cells reportedly program CD8+ T cells to develop into suitable memory cells during the primary immune response. However, new data indicate that the maintenance of CD8+ memory T cells requires continuous exposure to bystander CD4+ T cells.

Research paper thumbnail of CD4+CD25+Foxp3+ Regulatory T Cells Optimize Diversity of the Conventional T Cell Repertoire during Reconstitution from Lymphopenia

Journal of Immunology, May 1, 2010

Elevated IL-7 availability does not account for T cell proliferation in moderate lymphopenia. (10... more Elevated IL-7 availability does not account for T cell proliferation in moderate lymphopenia. (104.11)

Research paper thumbnail of Testing the susceptibility of mice with normal microbial experience to induction of allergic airway disease

Journal of Immunology, May 1, 2017

Allergic asthma is a large and growing medical problem in the developed world. The hygiene hypoth... more Allergic asthma is a large and growing medical problem in the developed world. The hygiene hypothesis posits that improved sanitation, vaccination programs, and increased antibiotic use have limited our exposure to natural infections, leading to immune systems that are more vulnerable to pathological responses. It has been difficult to test whether these advancements in public health can explain some of the increased incidence in allergic asthma, partly because laboratory mice commonly used in research are housed in a clean and highly controlled environment (known as specific pathogen free, or SPF). Cohousing SPF laboratory mice with mice from pet stores leads to acquisition of natural mouse pathogens through physiological animal-to-animal transmission. Our research group has demonstrated that this normal microbial experience dramatically alters the immune system, making it more similar to the immune system of adult humans. We used this “dirty” cohousing mouse model to test the effect of physiological microbial exposure on susceptibility to allergic airway disease. SPF and cohoused mice were sensitized and challenged intranasally with house dust mite (HDM) extract. Exposure to HDM induced eosinophilic airway inflammation and immune cell infiltration in SPF mice that resembles airway pathology of asthmatic patients. Serum levels of IgE and IgG1 antibodies to HDM were also elevated. Immune cell populations infiltrating the lung and activation of antigen-specific CD4+ T cells were also analyzed. We are currently investigating the immune response to this allergen exposure model in cohoused mice. Results from these studies will help explain the impact of environmental and microbial experience on susceptibility to allergic asthma.

Research paper thumbnail of The role of CD69 in generating resident memory CD8+ T cells

Journal of Immunology, May 1, 2016

Resident memory CD8+ T cells (TRM) are one of the first lines of defense against previously encou... more Resident memory CD8+ T cells (TRM) are one of the first lines of defense against previously encountered pathogens. Their advantageous position in parenchymal tissue combined with higher expression of effector molecules makes TRM particularly well suited to combat the early stages of an infection. CD69 is a canonical marker of typical CD8+ TRM. It has the potential to regulate TRM trafficking by competition with S1PR1 (and possibly other mechanisms). However, the functional requirement for CD69 to establish CD8+ TRM is unclear. To test this, we competed LCMV specific CD69 deficient and sufficient CD8+ T cells in vivo during the course of an acute LCMV infection. Unexpectedly, CD69 deficient cells were only weakly disadvantaged at establishing and maintaining residency in most parenchymal organs. However, CD69 deficient cells were heavily disadvantaged when establishing residency in the kidney. These insights lead to a more nuanced model of tissue residency, in which the requirement for CD69 expression may be dictated by factors such as the tissue microenvironment and antigen availability.

Research paper thumbnail of Cutting Edge: Krüppel-like Factor 2 Is Required for Phenotypic Maintenance but Not Development of B1 B Cells

Journal of Immunology, Oct 1, 2012

Krüppel-Like Factor 2 Regulates Tra cking and Homeostasis of γδ T Cells

Research paper thumbnail of NK Cell IL-10 Production Requires IL-15 and IL-10 Driven STAT3 Activation

Frontiers in Immunology, Sep 4, 2019

Natural killer (NK) cells can produce IFNγ or IL-10 to regulate inflammation and immune responses... more Natural killer (NK) cells can produce IFNγ or IL-10 to regulate inflammation and immune responses but the factors driving NK cell IL-10 secretion are poorly-defined. Here, we identified NK cell-intrinsic STAT3 activation as vital for IL-10 production during both systemic Listeria monocytogenes (Lm) infection and following IL-15 cytokine/receptor complex (IL15C) treatment for experimental cerebral malaria (ECM). In both contexts, conditional Stat3 deficiency in NK cells abrogated production of IL-10. Initial NK cell STAT3 phosphorylation was driven by IL-15. During Lm infection, this required capture or presentation of IL-15 by NK cell IL-15Rα. Persistent STAT3 activation was required to drive measurable IL-10 secretion and required NK cell expression of IL-10Rα. Survival-promoting effects of IL-15C treatment in ECM were dependent on NK cell Stat3 while NK cell-intrinsic deficiency for Stat3, Il15ra, or Il10ra abrogated NK cell IL-10 production and increased resistance against Lm. NK cell Stat3 deficiency did not impact production of IFNγ, indicating the STAT3 activation initiated by IL-15 and amplified by IL-10 selectively drives the production of anti-inflammatory IL-10 by responding NK cells.

Research paper thumbnail of Lung ILC2 responses to <i>Alternaria alternata</i> fungal allergen are blunted in “dirty” mice with physiologically transmitted murine microbes

Journal of Immunology, May 1, 2020

The objective of this study was to test the hypothesis that the increasing global incidence of al... more The objective of this study was to test the hypothesis that the increasing global incidence of allergy and atopy are due in part to improved hygiene and decreased microbial exposure. We have adopted a novel mouse model of normalized microbial exposure to test the impact of immune experience on subsequent responses to airway allergens. In this model, specific pathogen free (SPF) B6 mice are cohoused with mice from pet stores and become “dirty” – many commensals and pathogens are transmitted through cohousing and influence the immune cell populations systemically and in the lungs. We treated mice intranasally with a single dose of A. alternata fungal extract (Alt) and assessed production of type 2 cytokines. IL5 and IL13 levels in the lungs and bronchoalveolar lavage fluid were dramatically elevated by Alt in SPF mice but were not significantly increased by Alt in dirty mice. Type 2 innate lymphoid cells (ILC2) are the cells responsible for IL5 and IL13 after acute Alt treatment, and interestingly the number of lung ILC2 was unaltered by cohousing and their activation status appeared similar in both housing conditions. We treated mice with recombinant IL33, the alarmin released by lung epithelial cells in response to allergens, and the results suggest an impaired response to IL33 by dirty lung ILC2. In a repeated Alt exposure model, ILC2 cells expanded in SPF lungs and recruited eosinophils, neutrophils, and T cells. Most dirty mouse lungs contained these cell populations at steady state but were only modestly recruited with repeated Alt exposure. Lung function experiments are in progress. This study suggests that increased microbial exposure leads to more type-2 associated immune cells in the lungs, however responses to airway allergens are dampened.

Research paper thumbnail of Rapid cell cycling of both circulating and resident memory CD8+ T cells driven by common gamma chain-dependent cytokines

Journal of Immunology, May 1, 2021

Diverse populations of resident leukocytes live and self-renew within the tissues of the body, bu... more Diverse populations of resident leukocytes live and self-renew within the tissues of the body, but the mechanisms controlling their proliferation are poorly understood. Among these populations are many memory CD8+ T cells, which are found throughout the body and can locally control reinfection. A poor ability of vaccines to elicit stable and appropriate tissue-resident CD8+ T cell populations has likely contributed to long-standing challenges in vaccinating against several important pathogens. The factors required to support TRM proliferation in homeostasis are not well understood. We propose that proliferating TRMs flexibly use the available members of the common gamma chain-(γC)-dependent cytokine family to persist as bystanders during other immune responses. We have found that many γC-dependent cytokines can mediate cell-intrinsic signaling to drive proliferation of both circulating and resident memory CD8+ T cells of known antigen specificities without cognate antigen. Via transcriptional analysis of resting and in vivo cytokine-stimulated TRMs, we are elucidating downstream signaling and the gene expression signature of cytokine-driven TRM proliferation. We have also found that memory CD8+ T cells proliferate similarly in response to exogenous cytokines or unrelated pathogens, consistent with pathogen-elicited γC-dependent cytokine signals driving memory CD8+ T cell proliferation. These data support the concept that TRMs use γC-dependent cytokines produced during infections to support enhanced proliferation, likely to promote their maintenance. Our findings may lead to novel methods to therapeutically enhance TRM immunity.

Research paper thumbnail of IL-15 complex-stimulated NK cells protect mice from cerebral malaria

Journal of Immunology, May 1, 2016

Cerebral malaria (CM) is one of the most lethal complications of Plasmodium falciparum infection,... more Cerebral malaria (CM) is one of the most lethal complications of Plasmodium falciparum infection, responsible for a large fraction of the nearly one million malaria-related deaths annually. Infection of susceptible mouse strains such as C57BL/6 with Plasmodium berghei ANKA (PbA) induces a fatal neurological syndrome from 6–10 days post-infection (dpi) resulting from CD8 T cell-mediated damage to the CNS. We found that treatment of C57BL/6 mice with interleukin (IL)-15 complexes (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) prevented the development of PbA-induced CM. Rescue from CM was not associated with reduced parasitemia at 5 dpi. Instead, IL-15C treatment resulted in reduced CD8 T cell activation in the brain at 6 dpi and reduced blood brain barrier breakdown. In addition, adoptive transfer of IL-15C-stimulated natural killer (NK) cells (but not CD8+ T cells) was sufficient to prevent CM. IL-15C and similar complexes formed with IL-2 (IL-2C; IL-2 bound to the anti-IL-2 S4B6 antibody) both cause robust expansion and activation of NK cells but, unexpectedly, NK cells from mice treated with IL-2C failed to protect against CM. Comparative RNAseq analysis of IL-15C and IL-2C-treated NK cells identified novel gene expression patterns, demonstrating previously unappreciated differences between these cytokine complex signaling cascades in NK cells; the functional significance of these differences is being assessed. These data indicate that NK cells – which are typically involved in promoting inflammatory responses – can restrain damaging immune responses, and that differences between the activation of NK cells by IL-2C and IL-15C regulates their capacity to control the inflammatory response to blood stage malaria infection.

Research paper thumbnail of IL-4 sensitivity shapes the peripheral CD8+ T cell pool and response to infection

Journal of Immunology, May 1, 2016

Research paper thumbnail of Responsiveness to interleukin-15 therapy is shared between tissue-resident and circulating memory CD8 + T cell subsets

Proceedings of the National Academy of Sciences

Interleukin-15 (IL-15) is often considered a central regulator of memory CD8 + T cells, based pri... more Interleukin-15 (IL-15) is often considered a central regulator of memory CD8 + T cells, based primarily on studies of recirculating subsets. However, recent work identified IL-15–independent CD8 + T cell memory populations, including tissue-resident memory CD8 + T cells (T RM ) in some nonlymphoid tissues (NLTs). Whether this reflects the existence of IL-15–insensitive memory CD8 + T cells is unclear. We report that IL-15 complexes (IL-15c) stimulate rapid proliferation and expansion of both tissue-resident and circulating memory CD8 + T cell subsets across lymphoid and nonlymphoid tissues with varying magnitude by tissue and memory subset, in some sites correlating with differing levels of the IL-2Rβ. This was conserved for memory CD8 + T cells recognizing distinct antigens and elicited by different pathogens. Following IL-15c–induced expansion, divided cells contracted to baseline numbers and only slowly returned to basal proliferation, suggesting a mechanism to transiently amplif...

Research paper thumbnail of Defining the role of CD69 in the formation of resident memory CD8+ T cells

Journal of Immunology, 2018

Resident memory CD8+ T cells (T) reside in nonlymphoid tissues. There, they play a key role in pr... more Resident memory CD8+ T cells (T) reside in nonlymphoid tissues. There, they play a key role in preventing reinfection by exerting cytotoxic and inflammatory functions upon exposure to previously encountered pathogens. CD69 is often used as a definitive marker of T cells. CD69's interaction with the G-proteincoupled receptor S1PR1 has been identified as one mechanism by which CD69 can regulate tissue residency. However, the functional requirement for CD69 in promoting the generation and maintenance of CD8+ T under a wide variety of circumstances remains unclear. We explored the role of CD69 in tissue residency using co-transfer of antigen specific CD69 sufficient and deficient CD8+ T cells in the context of acute LCMV, Influenza, and VSV infections. Strikingly, we found that CD69 was not necessary for T establishment in most tissues, although it can promote T localization under some circumstances. This seems to be influenced by the focal point of infection. Interestingly, the kidney appears to rely on CD69 for tissue residency with every model pathogen examined. We propose that the requirement for CD69 is context dependent rather than absolute, and that a combination of factors, including tissue microenvironment and infectious agent, dictate CD69's influence on development of CD8+ resident memory.

Research paper thumbnail of Contributions of natural killer cells to the immune response against Plasmodium

Malaria Journal, 2019

Natural killer (NK) cells are important innate effector cells that are well described in their ab... more Natural killer (NK) cells are important innate effector cells that are well described in their ability to kill virally-infected cells and tumors. However, there is increasing appreciation for the role of NK cells in the control of other pathogens, including intracellular parasites such asPlasmodium, the cause of malaria. NK cells may be beneficial during the early phase ofPlasmodiuminfection—prior to the activation and expansion of antigen-specific T cells—through cooperation with myeloid cells to produce inflammatory cytokines like IFNγ. Recent work has defined howPlasmodiumcan activate NK cells to respond with natural cytotoxicity, and inhibit the growth of parasites via antibody-dependent cellular cytotoxicity mechanisms (ADCC). A specialized subset of adaptive NK cells that are negative for the Fc receptor γ chain have enhanced ADCC function and correlate with protection from malaria. Additionally, production of the regulatory cytokine IL-10 by NK cells prevents overt pathology ...

Research paper thumbnail of The Functional Requirement for CD69 in Establishment of Resident Memory CD8+ T Cells Varies with Tissue Location

The Journal of Immunology, 2019

Recent studies have characterized populations of memory CD8+ T cells that do not recirculate thro... more Recent studies have characterized populations of memory CD8+ T cells that do not recirculate through the blood but are, instead, retained in nonlymphoid tissues. Such CD8+ tissue resident memory T cells (TRM) are critical for pathogen control at barrier sites. Identifying TRM and defining the basis for their tissue residency is therefore of considerable importance for understanding protective immunity and improved vaccine design. Expression of the molecule CD69 is widely used as a definitive marker for TRM, yet it is unclear whether CD69 is universally required for producing or retaining TRM. Using multiple mouse models of acute immunization, we found that the functional requirement for CD69 was highly variable, depending on the tissue examined, playing no detectable role in generation of TRM at some sites (such as the small intestine), whereas CD69 was critical for establishing resident cells in the kidney. Likewise, forced expression of CD69 (but not expression of a CD69 mutant un...

Research paper thumbnail of Targeting glutamine metabolism rescues mice from late-stage cerebral malaria

Proceedings of the National Academy of Sciences of the United States of America, Jan 5, 2015

The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a c... more The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15-25% in African children despite effective antimalarial chemotherapy. There are no adjunctive treatments for CM, so there is an urgent need to identify new targets for therapy. Here we show that the glutamine analog 6-diazo-5-oxo-l-norleucine (DON) rescues mice from CM when administered late in the infection a time at which mice already are suffering blood-brain barrier dysfunction, brain swelling, and hemorrhaging accompanied by accumulation of parasite-specific CD8(+) effector T cells and infected red blood cells in the brain. Remarkably, within hours of DON treatment mice showed blood-brain barrier integrity, reduced brain swelling, decreased function of activated effector CD8(+) T cells in the brain, and levels of brain metabolites that resembled those in uninfected mice. These results suggest DON as a strong candidate for an effective adjunctive therapy for CM...

Research paper thumbnail of Neutrophil Involvement in Cross-Priming CD8+ T Cell Responses to Bacterial Antigens

The Journal of Immunology, 2004

Substantial CD8+ T cell responses are generated after infection of mice with recombinant Listeria... more Substantial CD8+ T cell responses are generated after infection of mice with recombinant Listeria monocytogenes strains expressing a model epitope (lymphocytic choriomeningitis virus NP118–126) in secreted and nonsecreted forms. L. monocytogenes gains access to the cytosol of infected cells, where secreted Ags can be accessed by the endogenous MHC class I presentation pathway. However, the route of presentation of the nonsecreted Ag in vivo remains undefined. In this study we show that neutrophil-enriched peritoneal exudate cells from L. monocytogenes-infected mice can serve as substrates for in vitro cross-presentation of both nonsecreted and secreted Ag by dendritic cells as well as for in vivo cross-priming of CD8+ T cells. In addition, specific neutrophil depletion in vivo by low dose treatment with either of two Ly6G-specific mAb substantially decreased the relative CD8+ T cell response against the nonsecreted, but not the secreted, Ag compared with control Ab-treated mice. Thu...

Research paper thumbnail of Quantitation of CD8+ T Cell Expansion, Memory, and Protective Immunity After Immunization with Peptide-Coated Dendritic Cells

The Journal of Immunology, 2002

Dendritic cells (DCs) are potent APCs for naive CD8+ T cells and are being investigated as vaccin... more Dendritic cells (DCs) are potent APCs for naive CD8+ T cells and are being investigated as vaccine delivery vehicles. In this study, we examine the CD8+ T cell response to defined peptides from Listeria monocytogenes (LM), lymphocytic choriomeningitis virus, and murine CMV coated singly and in combination onto mature bone marrow-derived DCs (BMDCs). We show that immunization of mice with 2 × 105 mature BMDCs coated with multiple MHC class I peptides generates a significant Ag-specific CD8+ T cell response in both the spleen and nonlymphoid organs. This immunization resulted in a peptide-specific hierarchy in the magnitude of CD8+ T cell priming and noncoordinate kinetics in response to different peptide epitopes. Kinetics were not exclusively due to specific characteristics of the MHC class I molecule, and were not altered in an Ag-independent manner by concurrent LM infection. Mice immunized with listeriolysin O 91–99-coated BMDCs are protected against high dose challenge with viru...

Research paper thumbnail of Listeria monocytogenesInfection Overcomes the Requirement for CD40 Ligand in Exogenous Antigen Presentation to CD8+ T Cells

The Journal of Immunology, 2001

In vivo priming of CD8+ T lymphocytes against exogenously processed model Ags requires CD4+ T cel... more In vivo priming of CD8+ T lymphocytes against exogenously processed model Ags requires CD4+ T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4+ T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8+ T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8+ T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8+ T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8+ T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immuniz...

Research paper thumbnail of Impaired Assembly yet Normal Trafficking of MHC Class I Molecules in Tapasin Mutant Mice

Immunity, 2000

sis, nascent class I heavy chains (HCs) associate with calnexin, an ER resident chaperone. The cl... more sis, nascent class I heavy chains (HCs) associate with calnexin, an ER resident chaperone. The class I HCs then bind with ␤ 2-microglobulin (␤ 2 m), and HC-␤ 2 m are

Research paper thumbnail of CD8+-T-Cell Response to Secreted and Nonsecreted Antigens Delivered by RecombinantListeria monocytogenesduring Secondary Infection

Infection and Immunity, 2002

ABSTRACTUnderstanding how existing antivector immunity impacts live vaccine delivery systems is c... more ABSTRACTUnderstanding how existing antivector immunity impacts live vaccine delivery systems is critical when the same vector system may be used to deliver different antigens. We addressed the impact of antivector immunity, elicited by immunization with attenuatedactA-deficientListeria monocytogenes, on the CD8+-T-cell response to a well-characterized lymphocytic choriomeningitis virus epitope, NP118-126, delivered by infection with recombinantL. monocytogenes. Challenges of immune mice withactA-deficient and with wild-type recombinantL. monocytogenesgenerated similar numbers of CD8+T cells specific for the NP118-126 epitope. High-dose immunization withactA-deficientL. monocytogenesresulted in substantial numbers of CD8+T cells specific for theL. monocytogenesLLO91-99 epitope in the effector and memory stages of the T-cell response. Challenge of these immune mice with recombinantL. monocytogenesresulted in rapid control of the infection and decreased CD8+-T-cell responses against bo...

Research paper thumbnail of Environmental conservation: bystander CD4 T cells keep CD8 memories fresh

Nature Immunology, Sep 1, 2004

CD4+ T cells reportedly program CD8+ T cells to develop into suitable memory cells during the pri... more CD4+ T cells reportedly program CD8+ T cells to develop into suitable memory cells during the primary immune response. However, new data indicate that the maintenance of CD8+ memory T cells requires continuous exposure to bystander CD4+ T cells.

Research paper thumbnail of CD4+CD25+Foxp3+ Regulatory T Cells Optimize Diversity of the Conventional T Cell Repertoire during Reconstitution from Lymphopenia

Journal of Immunology, May 1, 2010

Elevated IL-7 availability does not account for T cell proliferation in moderate lymphopenia. (10... more Elevated IL-7 availability does not account for T cell proliferation in moderate lymphopenia. (104.11)

Research paper thumbnail of Testing the susceptibility of mice with normal microbial experience to induction of allergic airway disease

Journal of Immunology, May 1, 2017

Allergic asthma is a large and growing medical problem in the developed world. The hygiene hypoth... more Allergic asthma is a large and growing medical problem in the developed world. The hygiene hypothesis posits that improved sanitation, vaccination programs, and increased antibiotic use have limited our exposure to natural infections, leading to immune systems that are more vulnerable to pathological responses. It has been difficult to test whether these advancements in public health can explain some of the increased incidence in allergic asthma, partly because laboratory mice commonly used in research are housed in a clean and highly controlled environment (known as specific pathogen free, or SPF). Cohousing SPF laboratory mice with mice from pet stores leads to acquisition of natural mouse pathogens through physiological animal-to-animal transmission. Our research group has demonstrated that this normal microbial experience dramatically alters the immune system, making it more similar to the immune system of adult humans. We used this “dirty” cohousing mouse model to test the effect of physiological microbial exposure on susceptibility to allergic airway disease. SPF and cohoused mice were sensitized and challenged intranasally with house dust mite (HDM) extract. Exposure to HDM induced eosinophilic airway inflammation and immune cell infiltration in SPF mice that resembles airway pathology of asthmatic patients. Serum levels of IgE and IgG1 antibodies to HDM were also elevated. Immune cell populations infiltrating the lung and activation of antigen-specific CD4+ T cells were also analyzed. We are currently investigating the immune response to this allergen exposure model in cohoused mice. Results from these studies will help explain the impact of environmental and microbial experience on susceptibility to allergic asthma.

Research paper thumbnail of The role of CD69 in generating resident memory CD8+ T cells

Journal of Immunology, May 1, 2016

Resident memory CD8+ T cells (TRM) are one of the first lines of defense against previously encou... more Resident memory CD8+ T cells (TRM) are one of the first lines of defense against previously encountered pathogens. Their advantageous position in parenchymal tissue combined with higher expression of effector molecules makes TRM particularly well suited to combat the early stages of an infection. CD69 is a canonical marker of typical CD8+ TRM. It has the potential to regulate TRM trafficking by competition with S1PR1 (and possibly other mechanisms). However, the functional requirement for CD69 to establish CD8+ TRM is unclear. To test this, we competed LCMV specific CD69 deficient and sufficient CD8+ T cells in vivo during the course of an acute LCMV infection. Unexpectedly, CD69 deficient cells were only weakly disadvantaged at establishing and maintaining residency in most parenchymal organs. However, CD69 deficient cells were heavily disadvantaged when establishing residency in the kidney. These insights lead to a more nuanced model of tissue residency, in which the requirement for CD69 expression may be dictated by factors such as the tissue microenvironment and antigen availability.

Research paper thumbnail of Cutting Edge: Krüppel-like Factor 2 Is Required for Phenotypic Maintenance but Not Development of B1 B Cells

Journal of Immunology, Oct 1, 2012

Krüppel-Like Factor 2 Regulates Tra cking and Homeostasis of γδ T Cells

Research paper thumbnail of NK Cell IL-10 Production Requires IL-15 and IL-10 Driven STAT3 Activation

Frontiers in Immunology, Sep 4, 2019

Natural killer (NK) cells can produce IFNγ or IL-10 to regulate inflammation and immune responses... more Natural killer (NK) cells can produce IFNγ or IL-10 to regulate inflammation and immune responses but the factors driving NK cell IL-10 secretion are poorly-defined. Here, we identified NK cell-intrinsic STAT3 activation as vital for IL-10 production during both systemic Listeria monocytogenes (Lm) infection and following IL-15 cytokine/receptor complex (IL15C) treatment for experimental cerebral malaria (ECM). In both contexts, conditional Stat3 deficiency in NK cells abrogated production of IL-10. Initial NK cell STAT3 phosphorylation was driven by IL-15. During Lm infection, this required capture or presentation of IL-15 by NK cell IL-15Rα. Persistent STAT3 activation was required to drive measurable IL-10 secretion and required NK cell expression of IL-10Rα. Survival-promoting effects of IL-15C treatment in ECM were dependent on NK cell Stat3 while NK cell-intrinsic deficiency for Stat3, Il15ra, or Il10ra abrogated NK cell IL-10 production and increased resistance against Lm. NK cell Stat3 deficiency did not impact production of IFNγ, indicating the STAT3 activation initiated by IL-15 and amplified by IL-10 selectively drives the production of anti-inflammatory IL-10 by responding NK cells.