Ag-specific memory T cell responses elicited by infections or vaccinations are inextricably linke... more Ag-specific memory T cell responses elicited by infections or vaccinations are inextricably linked to long-lasting protective immunity. Studies of protective immunity amongst residents of malaria endemic areas indicate that memory responses to Plasmodia antigens are not adequately developed or maintained. In contrast, multiple exposures to radiation-attenuated Plasmodia sporozoites (γ-spz) induce long-lasting protective immunity to experimental sporozoite challenge. In the P. berghei (Pb) γ-spz mouse model, lasting protection is associated with the presence of intrahepatic CD8 T cells that comprise two phenotypically and functionally discrete sets: effector/effector memory (TE/EM) cells that produce IFN-γ, and central memory (TCM) cells which express elevated levels of IL-15Rβ, suggesting that CD8 TCM cells depend upon IL-15 for maintenance and/or function. We asked whether immunization with Pb γ-spz could protect IL-15 KO mice against sporozoite challenge. The results demonstrate that although protective immunity is inducible in IL-15 KO mice, protection is short-lived, owing to reduced expression of Bcl-2 and increased apoptosis of proliferating intrahepatic CD8 TCM in the absence of IL-15. Therefore, we hypothesize that the maintenance of long-lasting protection induced by Pb γ-spz depends on a process whereby intrahepatic CD8 TCM cells, maintained by IL-15-mediated survival and basal proliferation, are conscripted into CD8 TE/EM cell pool during subsequent infections.
CD8 T cells specific for Plasmodium liver stage antigens (LS Ags) are considered key effectors in... more CD8 T cells specific for Plasmodium liver stage antigens (LS Ags) are considered key effectors in protective immunity induced by immunization with radiation-attenuated sporozoites (RAS). Sporozoite (spz)-stage epitopes that induce protective CD8 T cells have previously been identified, however, fine specificities of LS Ags-induced protective CD8 T cells remain unknown. Using transcriptome data based on LS forms of Plasmodium falciparum parasites, we identified several orthologue Plasmodium berghei (Pb) LS Ags that, as plasmid DNA vaccines delivered by Gene Gun immunization, significantly reduce liver parasite burden (LPB). Using algorithms that predict binding of peptides to H-2b alleles, we designed ~600 8–9 mer peptides derived from protective Pb LS Ags and screened them utilizing caged MHC class I-tetramer technology. Spleen cells from C57Bl/6 mice immunized thrice with Pb RAS and challenged with infectious spz were tested for binding to the conditional tetramers. We identified a...
A Plasmodium falciparum (Pf) genetically attenuated parasite (Pf GAP) vaccine, engineered by dele... more A Plasmodium falciparum (Pf) genetically attenuated parasite (Pf GAP) vaccine, engineered by deletion of 2 pre-erythrocytic genes, was administered to 6 subjects by bites from Anopheles mosquitoes. Previous studies of immune responses elicited by recombinant malaria vaccines show that protection is linked to Ag-specific T cells producing IFN-γ, TNF, and/or IL-2. In this study we asked if Pf GAP vaccine induced Ag-specific T cell responses characterized by inflammatory cytokines. We analyzed pre- and post vaccination PBMC for Ag-specific T cell responses to well characterized pre-erythrocytic and erythrocytic P. falciparum protein and peptide Ags. Additionally, we examined responses to several novel Pf liver-stage Ag (Pf LSA) because Pf LSA-dependent immunity might contribute to GAP-induced protection. IFN-γ and TNF responses to multiple antigens were significantly enhanced post vaccination; IFN-γ responses were primarily attributed to CD8+ T cells while TNF was secreted primarily by...
T lymphocytes are believed to play a major role in protection against malaria. Previous experimen... more T lymphocytes are believed to play a major role in protection against malaria. Previous experiments using in vivo depletion of CD8+ T cells, reconstitution with CD8+ T splenic cells, and adoptive transfer of CD8+ CTL clones demonstrated that protection against the exoerythrocytic stage of the murine strain, Plasmodium berghei malaria, was CD8+ T cell-dependent. Despite evidence for the critical role of CD8+ CTL, neither the cellular nor the molecular requirements for CD8+ T cell induction or for recognition of malaria Ags are known. In this study, we wished to define the role of CD8+ T cells and MHC class I molecules by using the P. berghei malaria attenuated sporozoites (SPZ) protection model in beta 2-microglobulin (beta 2m) knockout (-/-) mice. In contrast to observations that beta 2m-/- mice are resistant to many infectious diseases by compensatory mechanisms involving non-class I-restricted T cells, we found that beta 2m-/- mice failed to be protected against P. berghei SPZ, al...
<p>PBMC obtained at the indicated time points from RTS,S-immunized subjects were incubated ... more <p>PBMC obtained at the indicated time points from RTS,S-immunized subjects were incubated as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g002" target="_blank">Fig. 2</a> with CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cells were then analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>. The values are plotted as the frequency of IL-2-producing CD4<sup>+</sup> T<sub>CM</sub> cells (open bars) and IL-2-producing CD4<sup>+</sup> T<sub>E/EM</sub> cells (closed bars) for protected (right-hand panels) and non-protected (left-hand panels) subjects. Error bars indicate 95% confidence intervals. *p<0.05; **p<0.01; ***p<0.001.</p
<p>PBMC were obtained at the indicated time points from RTS,S-immunized subjects and were i... more <p>PBMC were obtained at the indicated time points from RTS,S-immunized subjects and were incubated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g003" target="_blank">Fig. 3</a> with CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cells were then analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>. The resulting values are plotted as the % of [A] IFN-γ producing CD4<sup>+</sup> T<sub>CM</sub> cells and [B] IFN-γ producing CD4<sup>+</sup> T<sub>E/EM</sub> cells for protected (closed symbol) and non-protected (open symbol) subjects. Statistically significant differences between pre-immune and post-vaccination time points are indicated at the top of the graph. Statistically significant differences between values for protected and non-protected subjects are as indicated within the body of the graph. *p<0.05; **p<0.01; ***p<0.001.</p
We conducted an open-label safety and immunogenicity bridging study that compared liquid and lyop... more We conducted an open-label safety and immunogenicity bridging study that compared liquid and lyophilized formulations of the candidate malaria vaccine RTS,S formulated in AS02A in 34 healthy, malaria-naïve adults at WRAIR. Volunteers received two doses of either formulation on a 0, 1-month schedule. Both vaccines were well tolerated and similarly immunogenic. Nineteen of 25 subjects who received the lyophilized formulation and six infectivity controls underwent sporozoite challenge to assess vaccine efficacy. All six controls had parasitemia detectable by thick blood smear by day 13 (mean pre-patent period 12.3 days; range 11-13). In the vaccine group, 8 of 19 vaccinees did not develop malaria and were completely protected (i.e., 42%). Among the 11 vaccinees who did become infected, the mean pre-patent period was delayed (14.4 days; range 13-18). The two formulations of RTS,S were equally safe and immunogenic, and the lyophilized formulation showed similar levels of efficacy against sporozoite challenge to that conferred by the liquid formulation in previous studies.
SummaryProtection induced by irradiated Plasmodium berghei sporozoites (Pbγ‐spz) in mice is linke... more SummaryProtection induced by irradiated Plasmodium berghei sporozoites (Pbγ‐spz) in mice is linked to CD8+ T cells specific for exo‐erythrocytic‐stage Ags, and intrahepatic memory CD8+ T cells are associated with protracted protection. However, the Ag specificity of the protective CD8+ T cells remains largely unknown. In this study, we characterized the TCR Vβ usage by intrahepatic CD8+ T cells during γ‐spz immunization and after the challenge with infectious Pb sporozoites. The repertoire of naïve (TN) and central memory (TCM) CD8+ T cells was diverse and conserved between individual mice, and did not change with immunization. In contrast, preferential usage of one or more TCR Vβ subset was observed in effector memory (TEM) CD8+ T cells after immunization. The expanded TCR Vβ varied between individual mice but Vβ4, 6, 7, 8.3, 9 and 11 were the most frequently expressed. In addition, there was a correlation in the TCR Vβ usage by γ‐spz‐induced CD8+ TEM in the liver and blood of indi...
Immunologic memory induced by pathogenic agents or vaccinations is inextricably linked to long-la... more Immunologic memory induced by pathogenic agents or vaccinations is inextricably linked to long-lasting protection. Adequately maintained memory T and B cell pools assure a fast, effective, and specific response against re-infections. Studies of immune responses amongst residents of malaria endemic areas suggest that memory responses to Plasmodia antigens appear to be neither adequately developed nor maintained, because persons who survive episodes of childhood malaria remain vulnerable to persistent or intermittent malaria infections. By contrast, multiple exposures of humans and laboratory rodents to radiation-attenuated Plasmodia sporozoites (γ-spz) induces sterile and long-lasting protection against experimental sporozoite challenge. Protection is associated with MHC-class I-dependent CD8T cells, the key effectors against pre-erythrocytic stage infection. We have adopted the P. berghei γ-spz mouse model to study memory CD8 T cells that are specific for antigens expressed by Pb liver-stage (LS) parasites and are found predominantly in the liver. On the basis of phenotypic and functional characteristics, we have demonstrated that liver CD8 T cells form two subsets: CD44 hi CD62L lo KLRG-1 + CD107 + CD127 − CD122 lo CD8 T effector/effector memory (T E/EM) cells that are the dominant IFN-γ producers and CD44 hi CD62L hi KLRG-1 − CD107 − CD127 + CD122 hi CD8T central memory (T CM) cells. In this review, we discuss our observations concerning the role of CD8 T E/EM and CD8 T CM cells in the maintenance of protracted protective immunity against experimental malaria infection. Finally, we present a hypothesis consistent with a model whereby intrahepatic CD8 T CM cells, that are maintained in part by LS-Ag depot and by IL-15-mediated survival and homeostatic proliferation, form a reservoir of cells ready for conscription to CD8T E/EM cells needed to prevent re-infections.
ABSTRACT We developed a minimalist cell-free antigen-processing system for MHC class II that can ... more ABSTRACT We developed a minimalist cell-free antigen-processing system for MHC class II that can identify physiologically selected T cell epitopes from antigens. The system utilizes purified proteins: HLA-DR1, cathepsin B, H, S, and HLA-DM. It was designed to mimic the specialized compartment for antigen processing with known defined molecular composition so that it can be a useful tool for elucidating steps involved in antigen processing and understanding the mechanisms of epitope selection. We found that prior digestion of antigens even for a few minutes eliminated the possibility of their immunodominant epitope capture by DR1. However, if those antigens were captured by DR1 first in the presence of DM, dominant epitopes were successfully selected. We observed simultaneously binding of DR1 and DR4 to denatured proteins detected by the biacore method. In another case, pre-digestion of antigen did not destroy the dominant epitope even though it showed sensitivity to DM mediated dissociation. We saw that this epitope got selected because it was less susceptible to enzymatic digestions and it was able to rebind to DR1 even in the presence of DM. Therefore, In general, most peptides generated from proteins do not get a chance to bind MHC II for presentation because they are sensitive to both DM and enzymatic digestions. Selection of immunodominant epitope is the results of collaborative reactions of DR1, DM, and cathepsins by increasing abundance of epitope capture by MHC II.
Induction of long-lasting protective immunity to malaria by repetitive immunization with P. bergh... more Induction of long-lasting protective immunity to malaria by repetitive immunization with P. berghei γ radiation-attenuated sporozoites (γ-spz) requires MHC class I-restricted CD8+ T cells, implying that γ-spz/liver stage derived antigens have to be presented through one of the cross-presentation pathways. Based on accumulation of proliferating and IFN-γ producing effector/memory CD8+ T cells in the livers of TAP−/− mice, we showed that TAP-independent vacuolar pathway efficiently operates in vivo during priming and subsequent boosts with γ-spz. Likewise, infectious sporozoite challenge increased the number of IFN-γ+ CD8+ T cells in γ-spz immune TAP−/− mice and induced IFN-γ production by wt γ-spz immune CD8+ T cells transferred into TAP−/− environment suggesting that γ-spz/liver stage derived MHC class I specific peptides generated in a TAP-independent manner participate in response to infectious sporozoites. Nevertheless, the pattern of IFN-γ secretion, parasitemia and survival dat...
Multiple exposures of humans and laboratory rodents to radiation-attenuated Plasmodium sporozoite... more Multiple exposures of humans and laboratory rodents to radiation-attenuated Plasmodium sporozoites (γ-spz) induce sterile and long-lasting protection that is thought to be directed mainly to liver-stage antigens (LS-Ags). Although protection involves B cells and CD4 T cells, MHC class I-dependent liver IFN-γ+ CD8 T cells are the key effectors against LS-Ags. Recently several LS-Ags have been shown to induce protection in murine models; nevertheless, the mechanism of processing and presentation of LS-Ag to CD8 T cells has not been investigated. We tested liver CD8 T cell responses induced by Plasmodium berghei (Pb) γ-spz in TAP-/- mice to inquire whether processing/presentation of the exogenous LS-Ags was TAP-dependent or -independent. Immunization with Pb γ-spz induced proliferation and IFN-γ production by liver CD8 T cells. Also, TAP-/- CD8 T cells eliminated hepatocytes infected with Pb sporozoites in vitro. Despite these effector activities, Pb γ-spz immunized TAP-/- mice were no...
Plasmodium berghei sporozoite (SPZ)-immune lymph node (LN) cells obtained from mice of different ... more Plasmodium berghei sporozoite (SPZ)-immune lymph node (LN) cells obtained from mice of different H-2 haplotypes were analyzed for the presence of circumsporozoite (CS) protein-reactive T cells in proliferative assays. Although lymphocytes from each strain responded in vitro to the priming Ag and to the soluble rCS protein, they did not respond to CS protein synthetic peptides. Parallel analysis of rCS protein-primed LN cells revealed that the two Ag are unequal in generating T cell specificities: although SPZ priming did not induce CS protein peptide-reactive T cells, priming with rCS protein did. Not being privy to the processing and presentation of SPZ Ag, we postulated that a different order of processing of the authentic, i.e., SPZ-associated CS protein vs soluble rCS protein might be responsible for the generation of different T cell specificities. Accordingly, authentic CS protein might not be processed by APC, or the processed fragments might obscure the recognition of smalle...
The fine specificity of the T cell repertoire directed against T helper (Th)-inducing and T suppr... more The fine specificity of the T cell repertoire directed against T helper (Th)-inducing and T suppressor (Ts)-529-591; 1985.
Ag-specific memory T cell responses elicited by infections or vaccinations are inextricably linke... more Ag-specific memory T cell responses elicited by infections or vaccinations are inextricably linked to long-lasting protective immunity. Studies of protective immunity amongst residents of malaria endemic areas indicate that memory responses to Plasmodia antigens are not adequately developed or maintained. In contrast, multiple exposures to radiation-attenuated Plasmodia sporozoites (γ-spz) induce long-lasting protective immunity to experimental sporozoite challenge. In the P. berghei (Pb) γ-spz mouse model, lasting protection is associated with the presence of intrahepatic CD8 T cells that comprise two phenotypically and functionally discrete sets: effector/effector memory (TE/EM) cells that produce IFN-γ, and central memory (TCM) cells which express elevated levels of IL-15Rβ, suggesting that CD8 TCM cells depend upon IL-15 for maintenance and/or function. We asked whether immunization with Pb γ-spz could protect IL-15 KO mice against sporozoite challenge. The results demonstrate that although protective immunity is inducible in IL-15 KO mice, protection is short-lived, owing to reduced expression of Bcl-2 and increased apoptosis of proliferating intrahepatic CD8 TCM in the absence of IL-15. Therefore, we hypothesize that the maintenance of long-lasting protection induced by Pb γ-spz depends on a process whereby intrahepatic CD8 TCM cells, maintained by IL-15-mediated survival and basal proliferation, are conscripted into CD8 TE/EM cell pool during subsequent infections.
CD8 T cells specific for Plasmodium liver stage antigens (LS Ags) are considered key effectors in... more CD8 T cells specific for Plasmodium liver stage antigens (LS Ags) are considered key effectors in protective immunity induced by immunization with radiation-attenuated sporozoites (RAS). Sporozoite (spz)-stage epitopes that induce protective CD8 T cells have previously been identified, however, fine specificities of LS Ags-induced protective CD8 T cells remain unknown. Using transcriptome data based on LS forms of Plasmodium falciparum parasites, we identified several orthologue Plasmodium berghei (Pb) LS Ags that, as plasmid DNA vaccines delivered by Gene Gun immunization, significantly reduce liver parasite burden (LPB). Using algorithms that predict binding of peptides to H-2b alleles, we designed ~600 8–9 mer peptides derived from protective Pb LS Ags and screened them utilizing caged MHC class I-tetramer technology. Spleen cells from C57Bl/6 mice immunized thrice with Pb RAS and challenged with infectious spz were tested for binding to the conditional tetramers. We identified a...
A Plasmodium falciparum (Pf) genetically attenuated parasite (Pf GAP) vaccine, engineered by dele... more A Plasmodium falciparum (Pf) genetically attenuated parasite (Pf GAP) vaccine, engineered by deletion of 2 pre-erythrocytic genes, was administered to 6 subjects by bites from Anopheles mosquitoes. Previous studies of immune responses elicited by recombinant malaria vaccines show that protection is linked to Ag-specific T cells producing IFN-γ, TNF, and/or IL-2. In this study we asked if Pf GAP vaccine induced Ag-specific T cell responses characterized by inflammatory cytokines. We analyzed pre- and post vaccination PBMC for Ag-specific T cell responses to well characterized pre-erythrocytic and erythrocytic P. falciparum protein and peptide Ags. Additionally, we examined responses to several novel Pf liver-stage Ag (Pf LSA) because Pf LSA-dependent immunity might contribute to GAP-induced protection. IFN-γ and TNF responses to multiple antigens were significantly enhanced post vaccination; IFN-γ responses were primarily attributed to CD8+ T cells while TNF was secreted primarily by...
T lymphocytes are believed to play a major role in protection against malaria. Previous experimen... more T lymphocytes are believed to play a major role in protection against malaria. Previous experiments using in vivo depletion of CD8+ T cells, reconstitution with CD8+ T splenic cells, and adoptive transfer of CD8+ CTL clones demonstrated that protection against the exoerythrocytic stage of the murine strain, Plasmodium berghei malaria, was CD8+ T cell-dependent. Despite evidence for the critical role of CD8+ CTL, neither the cellular nor the molecular requirements for CD8+ T cell induction or for recognition of malaria Ags are known. In this study, we wished to define the role of CD8+ T cells and MHC class I molecules by using the P. berghei malaria attenuated sporozoites (SPZ) protection model in beta 2-microglobulin (beta 2m) knockout (-/-) mice. In contrast to observations that beta 2m-/- mice are resistant to many infectious diseases by compensatory mechanisms involving non-class I-restricted T cells, we found that beta 2m-/- mice failed to be protected against P. berghei SPZ, al...
<p>PBMC obtained at the indicated time points from RTS,S-immunized subjects were incubated ... more <p>PBMC obtained at the indicated time points from RTS,S-immunized subjects were incubated as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g002" target="_blank">Fig. 2</a> with CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cells were then analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>. The values are plotted as the frequency of IL-2-producing CD4<sup>+</sup> T<sub>CM</sub> cells (open bars) and IL-2-producing CD4<sup>+</sup> T<sub>E/EM</sub> cells (closed bars) for protected (right-hand panels) and non-protected (left-hand panels) subjects. Error bars indicate 95% confidence intervals. *p<0.05; **p<0.01; ***p<0.001.</p
<p>PBMC were obtained at the indicated time points from RTS,S-immunized subjects and were i... more <p>PBMC were obtained at the indicated time points from RTS,S-immunized subjects and were incubated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g003" target="_blank">Fig. 3</a> with CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cells were then analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>. The resulting values are plotted as the % of [A] IFN-γ producing CD4<sup>+</sup> T<sub>CM</sub> cells and [B] IFN-γ producing CD4<sup>+</sup> T<sub>E/EM</sub> cells for protected (closed symbol) and non-protected (open symbol) subjects. Statistically significant differences between pre-immune and post-vaccination time points are indicated at the top of the graph. Statistically significant differences between values for protected and non-protected subjects are as indicated within the body of the graph. *p<0.05; **p<0.01; ***p<0.001.</p
We conducted an open-label safety and immunogenicity bridging study that compared liquid and lyop... more We conducted an open-label safety and immunogenicity bridging study that compared liquid and lyophilized formulations of the candidate malaria vaccine RTS,S formulated in AS02A in 34 healthy, malaria-naïve adults at WRAIR. Volunteers received two doses of either formulation on a 0, 1-month schedule. Both vaccines were well tolerated and similarly immunogenic. Nineteen of 25 subjects who received the lyophilized formulation and six infectivity controls underwent sporozoite challenge to assess vaccine efficacy. All six controls had parasitemia detectable by thick blood smear by day 13 (mean pre-patent period 12.3 days; range 11-13). In the vaccine group, 8 of 19 vaccinees did not develop malaria and were completely protected (i.e., 42%). Among the 11 vaccinees who did become infected, the mean pre-patent period was delayed (14.4 days; range 13-18). The two formulations of RTS,S were equally safe and immunogenic, and the lyophilized formulation showed similar levels of efficacy against sporozoite challenge to that conferred by the liquid formulation in previous studies.
SummaryProtection induced by irradiated Plasmodium berghei sporozoites (Pbγ‐spz) in mice is linke... more SummaryProtection induced by irradiated Plasmodium berghei sporozoites (Pbγ‐spz) in mice is linked to CD8+ T cells specific for exo‐erythrocytic‐stage Ags, and intrahepatic memory CD8+ T cells are associated with protracted protection. However, the Ag specificity of the protective CD8+ T cells remains largely unknown. In this study, we characterized the TCR Vβ usage by intrahepatic CD8+ T cells during γ‐spz immunization and after the challenge with infectious Pb sporozoites. The repertoire of naïve (TN) and central memory (TCM) CD8+ T cells was diverse and conserved between individual mice, and did not change with immunization. In contrast, preferential usage of one or more TCR Vβ subset was observed in effector memory (TEM) CD8+ T cells after immunization. The expanded TCR Vβ varied between individual mice but Vβ4, 6, 7, 8.3, 9 and 11 were the most frequently expressed. In addition, there was a correlation in the TCR Vβ usage by γ‐spz‐induced CD8+ TEM in the liver and blood of indi...
Immunologic memory induced by pathogenic agents or vaccinations is inextricably linked to long-la... more Immunologic memory induced by pathogenic agents or vaccinations is inextricably linked to long-lasting protection. Adequately maintained memory T and B cell pools assure a fast, effective, and specific response against re-infections. Studies of immune responses amongst residents of malaria endemic areas suggest that memory responses to Plasmodia antigens appear to be neither adequately developed nor maintained, because persons who survive episodes of childhood malaria remain vulnerable to persistent or intermittent malaria infections. By contrast, multiple exposures of humans and laboratory rodents to radiation-attenuated Plasmodia sporozoites (γ-spz) induces sterile and long-lasting protection against experimental sporozoite challenge. Protection is associated with MHC-class I-dependent CD8T cells, the key effectors against pre-erythrocytic stage infection. We have adopted the P. berghei γ-spz mouse model to study memory CD8 T cells that are specific for antigens expressed by Pb liver-stage (LS) parasites and are found predominantly in the liver. On the basis of phenotypic and functional characteristics, we have demonstrated that liver CD8 T cells form two subsets: CD44 hi CD62L lo KLRG-1 + CD107 + CD127 − CD122 lo CD8 T effector/effector memory (T E/EM) cells that are the dominant IFN-γ producers and CD44 hi CD62L hi KLRG-1 − CD107 − CD127 + CD122 hi CD8T central memory (T CM) cells. In this review, we discuss our observations concerning the role of CD8 T E/EM and CD8 T CM cells in the maintenance of protracted protective immunity against experimental malaria infection. Finally, we present a hypothesis consistent with a model whereby intrahepatic CD8 T CM cells, that are maintained in part by LS-Ag depot and by IL-15-mediated survival and homeostatic proliferation, form a reservoir of cells ready for conscription to CD8T E/EM cells needed to prevent re-infections.
ABSTRACT We developed a minimalist cell-free antigen-processing system for MHC class II that can ... more ABSTRACT We developed a minimalist cell-free antigen-processing system for MHC class II that can identify physiologically selected T cell epitopes from antigens. The system utilizes purified proteins: HLA-DR1, cathepsin B, H, S, and HLA-DM. It was designed to mimic the specialized compartment for antigen processing with known defined molecular composition so that it can be a useful tool for elucidating steps involved in antigen processing and understanding the mechanisms of epitope selection. We found that prior digestion of antigens even for a few minutes eliminated the possibility of their immunodominant epitope capture by DR1. However, if those antigens were captured by DR1 first in the presence of DM, dominant epitopes were successfully selected. We observed simultaneously binding of DR1 and DR4 to denatured proteins detected by the biacore method. In another case, pre-digestion of antigen did not destroy the dominant epitope even though it showed sensitivity to DM mediated dissociation. We saw that this epitope got selected because it was less susceptible to enzymatic digestions and it was able to rebind to DR1 even in the presence of DM. Therefore, In general, most peptides generated from proteins do not get a chance to bind MHC II for presentation because they are sensitive to both DM and enzymatic digestions. Selection of immunodominant epitope is the results of collaborative reactions of DR1, DM, and cathepsins by increasing abundance of epitope capture by MHC II.
Induction of long-lasting protective immunity to malaria by repetitive immunization with P. bergh... more Induction of long-lasting protective immunity to malaria by repetitive immunization with P. berghei γ radiation-attenuated sporozoites (γ-spz) requires MHC class I-restricted CD8+ T cells, implying that γ-spz/liver stage derived antigens have to be presented through one of the cross-presentation pathways. Based on accumulation of proliferating and IFN-γ producing effector/memory CD8+ T cells in the livers of TAP−/− mice, we showed that TAP-independent vacuolar pathway efficiently operates in vivo during priming and subsequent boosts with γ-spz. Likewise, infectious sporozoite challenge increased the number of IFN-γ+ CD8+ T cells in γ-spz immune TAP−/− mice and induced IFN-γ production by wt γ-spz immune CD8+ T cells transferred into TAP−/− environment suggesting that γ-spz/liver stage derived MHC class I specific peptides generated in a TAP-independent manner participate in response to infectious sporozoites. Nevertheless, the pattern of IFN-γ secretion, parasitemia and survival dat...
Multiple exposures of humans and laboratory rodents to radiation-attenuated Plasmodium sporozoite... more Multiple exposures of humans and laboratory rodents to radiation-attenuated Plasmodium sporozoites (γ-spz) induce sterile and long-lasting protection that is thought to be directed mainly to liver-stage antigens (LS-Ags). Although protection involves B cells and CD4 T cells, MHC class I-dependent liver IFN-γ+ CD8 T cells are the key effectors against LS-Ags. Recently several LS-Ags have been shown to induce protection in murine models; nevertheless, the mechanism of processing and presentation of LS-Ag to CD8 T cells has not been investigated. We tested liver CD8 T cell responses induced by Plasmodium berghei (Pb) γ-spz in TAP-/- mice to inquire whether processing/presentation of the exogenous LS-Ags was TAP-dependent or -independent. Immunization with Pb γ-spz induced proliferation and IFN-γ production by liver CD8 T cells. Also, TAP-/- CD8 T cells eliminated hepatocytes infected with Pb sporozoites in vitro. Despite these effector activities, Pb γ-spz immunized TAP-/- mice were no...
Plasmodium berghei sporozoite (SPZ)-immune lymph node (LN) cells obtained from mice of different ... more Plasmodium berghei sporozoite (SPZ)-immune lymph node (LN) cells obtained from mice of different H-2 haplotypes were analyzed for the presence of circumsporozoite (CS) protein-reactive T cells in proliferative assays. Although lymphocytes from each strain responded in vitro to the priming Ag and to the soluble rCS protein, they did not respond to CS protein synthetic peptides. Parallel analysis of rCS protein-primed LN cells revealed that the two Ag are unequal in generating T cell specificities: although SPZ priming did not induce CS protein peptide-reactive T cells, priming with rCS protein did. Not being privy to the processing and presentation of SPZ Ag, we postulated that a different order of processing of the authentic, i.e., SPZ-associated CS protein vs soluble rCS protein might be responsible for the generation of different T cell specificities. Accordingly, authentic CS protein might not be processed by APC, or the processed fragments might obscure the recognition of smalle...
The fine specificity of the T cell repertoire directed against T helper (Th)-inducing and T suppr... more The fine specificity of the T cell repertoire directed against T helper (Th)-inducing and T suppressor (Ts)-529-591; 1985.
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Papers by Urszula Krzych