I. Munitic - Academia.edu (original) (raw)

Papers by I. Munitic

Research paper thumbnail of  c deficiency precludes CD8+ T cell memory despite formation of potent T cell effectors

Proceedings of the National Academy of Sciences, 2010

Several cytokines (including IL-2, IL-7, IL-15, and IL-21) that signal through receptors sharing ... more Several cytokines (including IL-2, IL-7, IL-15, and IL-21) that signal through receptors sharing the common γ chain (γ c ) are critical for the generation and peripheral homeostasis of naive and memory T cells. Recently, we demonstrated that effector functions fail to develop in CD4 + T cells that differentiate in the absence of γ c . To assess the role of γ c cytokines in cell-fate decisions that condition effector versus memory CD8 + T cell generation, we compared the response of CD8 + T cells from γ c + or γ c − P14 TCR transgenic mice after challenge with lymphocytic choriomeningitis virus. The intrinsic IL-7-dependent survival defect of γ c − naive CD8 + T cells was corrected by transgenic expression of human Bcl-2. We demonstrated that although γ c -dependent signals are dispensable for the initial expansion and the acquisition of cytotoxic functions following antigenic stimulation, they condition the terminal proliferation and differentiation of CD8 + effector T cells (i.e., ...

Research paper thumbnail of CD70 Deficiency Impairs Effector CD8 T Cell Generation and Viral Clearance but Is Dispensable for the Recall Response to Lymphocytic Choriomeningitis Virus

The Journal of Immunology, 2012

CD27 interactions with its ligand, CD70, are thought to be necessary for optimal primary and memo... more CD27 interactions with its ligand, CD70, are thought to be necessary for optimal primary and memory adaptive immune responses to a variety of pathogens. Thus far, all studies addressing the function of the CD27-CD70 axis have been performed in mice lacking CD27, in those overexpressing CD70, or in those in which these molecules were blocked or mimicked by Abs or recombinant soluble CD70. Because these methods have in some cases led to divergent results, we generated CD70-deficient mice to directly assess its role in vivo. We find that lack of CD70-mediated stimulation during primary responses to lymphocytic choriomeningitis virus lowered the magnitude of CD8 Ag-specific T cell response, resulting in impaired viral clearance, without affecting CD4 T cell responses. Unexpectedly, CD70-CD27 costimulation was not needed for memory CD8 T cell generation or the ability to mount a recall response to lymphocytic choriomeningitis virus. Adoptive transfers of wild-type memory T cells into CD70 2/2 or wildtype hosts also showed no need for CD70-mediated stimulation during the course of the recall response. Moreover, CD70 expression by CD8 T cells could not rescue endogenous CD70 2/2 cells from defective expansion, arguing against a role for CD70-mediated T:T help in this model. Therefore, CD70 appears to be an important factor in the initiation of a robust and effective primary response but dispensable for CD8 T cell memory responses.

Research paper thumbnail of Epitope Specificity and Relative Clonal Abundance Do Not Affect CD8 Differentiation Patterns during Lymphocytic Choriomeningitis Virus Infection

Journal of Virology, 2009

To evaluate the impact of immunodominance on CD8 T-cell properties, we compared the functional pr... more To evaluate the impact of immunodominance on CD8 T-cell properties, we compared the functional properties of dominant and subdominant populations in the response to lymphocytic choriomeningitis virus (LCMV). To improve functional discrimination, in addition to the usual tests of phenotype and function, we used a sensitive technique that allows the screening of all CD8 effector genes simultaneously in single cells. Surprisingly, these methods failed to reveal a major impact of clonal dominance in CD8 properties throughout the response. Aiming to increase clonal dominance, we examined high-frequency transferred P14 T-cell receptor transgenic (TCR Tg) cells. Under these conditions LCMV is cleared faster, and accordingly we found an accelerated response. However, when Tg and endogenous cells were studied in the same mice, where they should be subjected to the same antigen load, they showed overlapping properties, and the presence of P14 cells did not modify endogenous responses to other...

Research paper thumbnail of Optineurin Insufficiency Impairs IRF3 but Not NF- B Activation in Immune Cells

The Journal of Immunology, 2013

Research paper thumbnail of T Cells in G1 Provide a Memory-Like Response to Secondary Stimulation

The Journal of Immunology, 2005

The commitment of naive T cells to proliferate is a function of the strength and duration of stim... more The commitment of naive T cells to proliferate is a function of the strength and duration of stimuli mediated by the TCR and coreceptors. Ranges of 2-20 h of stimulation have been reported as necessary in vitro. Whether T cells actually experience uninterrupted stimulation for such long periods under physiological conditions is controversial. Here we ask whether commitment to proliferate requires continuous stimulation, or can T cells integrate intermittent periods of stimulation. T cells were stimulated for two short-term (subthreshold) periods (5-7 h) either sequentially or separated by an interval of rest. Naive lymph node T cells were able to integrate interrupted stimulation, even when the duration of rest was as long as 2 days. Furthermore, when shortterm-stimulated T cells were separated by density, three populations were observed: low density blasts, intermediate density G 1 cells, and high density G 0 cells. Low density cells progressed to division without further stimulation, whereas G 0 and G 1 cells remained undivided. However, after a period of rest, a second subthreshold stimulation caused the G 1 but not the G 0 fraction to quickly proceed through the cell cycle. We conclude that noncycling T cells in the G 1 phase of the cell cycle remain in a state of readiness for prolonged periods of time, and may represent a population of memory-like effectors capable of responding rapidly to antigenic challenge.

Research paper thumbnail of Regulation of Constitutive TCR Internalization by the  -Chain

The Journal of Immunology, 2002

The ability of a T cell to be activated is critically regulated by the number of TCRs expressed o... more The ability of a T cell to be activated is critically regulated by the number of TCRs expressed on the plasma membrane. Cell surface TCR expression is influenced by dynamic processes such as synthesis and transport of newly assembled receptors, endocytosis of surface TCR, and recycling to the plasma membrane of internalized receptors. In this study, the internalization of fluorescently labeled anti-TCR Abs was used to analyze constitutive endocytosis of TCRs on T cells, and to investigate the role of the zeta-chain in this process. We found that cell surface TCRs lacking zeta were endocytosed more rapidly than completely assembled receptors, and that reexpression of full-length zeta led to a dose-dependent decrease in the rate of TCR internalization. Rapid TCR internalization was also observed with CD4(+)CD8(+) thymocytes from zeta-deficient mice, whereas TCR internalization on thymocytes from CD3-delta deficient animals was slow, similar to that of wild-type thymocytes. This identifies a specific role for zeta in the regulation of constitutive receptor internalization. Furthermore, chimeric zeta molecules containing non-native intracellular amino acid sequences also led to high levels of TCR expression and reduced TCR cycling. These effects were dependent solely on the length of the intracellular tail, ruling out a role for intracellular zeta-specific interactions with other molecules as a mechanism for regulating TCR internalization. Rather, these findings strongly support a model in which the zeta-chain stabilizes TCR residency on the cell surface, and functions to maintain cell surface receptor expression by sterically blocking internalization sequences in other TCR components.

Research paper thumbnail of CD70 Is Downregulated by Interaction with CD27

The Journal of Immunology, 2013

Research paper thumbnail of CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response

Journal of Experimental Medicine, 2007

Research paper thumbnail of The CD8+ memory T-cell state of readiness is actively maintained and reversible

Blood, 2009

The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen e... more The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G(1) to the G(0) cell-cycle state and responded to stimulation like naive T cells, as assessed by proliferation, dependence upon costimulation, and interferon-gamma production, without losing cell surface markers associated with memory. The memory state was maintained by signaling via members of the tumor necrosis factor receptor superfamily, CD27 and 4-1BB. Foxo1, a transcription factor involved in T-cell quiescence, was reduced in memory cells, and stimulation of naive CD8 cells via CD27 caused Foxo1 to be phosphorylated and emigrate from the nucleus in a phosphatidylinositol-3 kinase-dependent manner. Consistent with these results, maintenance of G(1) in vivo was compromised in antigen-specific memory T cells in vesicular stomatitis virus-infected CD27-deficient mice. Therefore, sustaining the functional phenotype of T memory cells requires active signaling and maintenance.

Research paper thumbnail of Dynamic regulation of IL-7 receptor expression is required for normal thymopoiesis

Blood, 2004

IL-7 receptor (IL-7R) levels are tightly controlled during ontogeny: high on DN cells, absent on ... more IL-7 receptor (IL-7R) levels are tightly controlled during ontogeny: high on DN cells, absent on DP cells, and high once again on thymocytes undergoing positive selection. To determine if loss of IL-7-mediated survival signals in DP cells is necessary for normal antigen-specific selection, we created T-lineage specific IL-7R α chain (IL-7Rα) transgenic (Tg) mice in which IL-7R is expressed throughout ontogeny. There was no effect of the IL-7Rα Tg on negative selection. Surprisingly, however, although the thymi of IL-7Rα Tg mice were comparable at birth, there was a decrease in thymocyte number as the mice aged. This was found to be due to competition between DN and IL-7Rexpressing DP cells for endogenous IL-7, which resulted in decreased levels of Bcl-2 in DN cells, increased DN apoptosis, and decreased DN cell number. Therefore, the downregulation of IL-7R on DP cells is an "altruistic" act required for maintaining an adequate supply of local IL-7 for DN cells.

Research paper thumbnail of Epitope Specificity and Relative Clonal Abundance Do Not Affect CD8 Differentiation Patterns during Lymphocytic Choriomeningitis Virus Infection

Journal of Virology, 2009

To evaluate the impact of immunodominance on CD8 T-cell properties, we compared the functional pr... more To evaluate the impact of immunodominance on CD8 T-cell properties, we compared the functional properties of dominant and subdominant populations in the response to lymphocytic choriomeningitis virus (LCMV). To improve functional discrimination, in addition to the usual tests of phenotype and function, we used a sensitive technique that allows the screening of all CD8 effector genes simultaneously in single cells. Surprisingly, these methods failed to reveal a major impact of clonal dominance in CD8 properties throughout the response. Aiming to increase clonal dominance, we examined high-frequency transferred P14 T-cell receptor transgenic (TCR Tg) cells. Under these conditions LCMV is cleared faster, and accordingly we found an accelerated response. However, when Tg and endogenous cells were studied in the same mice, where they should be subjected to the same antigen load, they showed overlapping properties, and the presence of P14 cells did not modify endogenous responses to other LCMV epitopes or a perturbed immunodominance hierarchy in the memory phase. Using allotype-labeled Tg cells, we found that during acute infection up to 80% downregulated their TCR and were undetectable by tetramer binding, and that tetramer-negative and tetramer-positive cells had very different features. Since Tg cells are not available to evaluate immune responses in humans and, in many cases, are not available from the mouse, the tetramer-based evaluation of early immune responses in most situations of high viremia may be incomplete and biased.

Research paper thumbnail of  c deficiency precludes CD8+ T cell memory despite formation of potent T cell effectors

Proceedings of the National Academy of Sciences, 2010

Several cytokines (including IL-2, IL-7, IL-15, and IL-21) that signal through receptors sharing ... more Several cytokines (including IL-2, IL-7, IL-15, and IL-21) that signal through receptors sharing the common γ chain (γ c ) are critical for the generation and peripheral homeostasis of naive and memory T cells. Recently, we demonstrated that effector functions fail to develop in CD4 + T cells that differentiate in the absence of γ c . To assess the role of γ c cytokines in cell-fate decisions that condition effector versus memory CD8 + T cell generation, we compared the response of CD8 + T cells from γ c + or γ c − P14 TCR transgenic mice after challenge with lymphocytic choriomeningitis virus. The intrinsic IL-7-dependent survival defect of γ c − naive CD8 + T cells was corrected by transgenic expression of human Bcl-2. We demonstrated that although γ c -dependent signals are dispensable for the initial expansion and the acquisition of cytotoxic functions following antigenic stimulation, they condition the terminal proliferation and differentiation of CD8 + effector T cells (i.e., ...

Research paper thumbnail of CD70 Deficiency Impairs Effector CD8 T Cell Generation and Viral Clearance but Is Dispensable for the Recall Response to Lymphocytic Choriomeningitis Virus

The Journal of Immunology, 2012

CD27 interactions with its ligand, CD70, are thought to be necessary for optimal primary and memo... more CD27 interactions with its ligand, CD70, are thought to be necessary for optimal primary and memory adaptive immune responses to a variety of pathogens. Thus far, all studies addressing the function of the CD27-CD70 axis have been performed in mice lacking CD27, in those overexpressing CD70, or in those in which these molecules were blocked or mimicked by Abs or recombinant soluble CD70. Because these methods have in some cases led to divergent results, we generated CD70-deficient mice to directly assess its role in vivo. We find that lack of CD70-mediated stimulation during primary responses to lymphocytic choriomeningitis virus lowered the magnitude of CD8 Ag-specific T cell response, resulting in impaired viral clearance, without affecting CD4 T cell responses. Unexpectedly, CD70-CD27 costimulation was not needed for memory CD8 T cell generation or the ability to mount a recall response to lymphocytic choriomeningitis virus. Adoptive transfers of wild-type memory T cells into CD70 2/2 or wildtype hosts also showed no need for CD70-mediated stimulation during the course of the recall response. Moreover, CD70 expression by CD8 T cells could not rescue endogenous CD70 2/2 cells from defective expansion, arguing against a role for CD70-mediated T:T help in this model. Therefore, CD70 appears to be an important factor in the initiation of a robust and effective primary response but dispensable for CD8 T cell memory responses.

Research paper thumbnail of Epitope Specificity and Relative Clonal Abundance Do Not Affect CD8 Differentiation Patterns during Lymphocytic Choriomeningitis Virus Infection

Journal of Virology, 2009

To evaluate the impact of immunodominance on CD8 T-cell properties, we compared the functional pr... more To evaluate the impact of immunodominance on CD8 T-cell properties, we compared the functional properties of dominant and subdominant populations in the response to lymphocytic choriomeningitis virus (LCMV). To improve functional discrimination, in addition to the usual tests of phenotype and function, we used a sensitive technique that allows the screening of all CD8 effector genes simultaneously in single cells. Surprisingly, these methods failed to reveal a major impact of clonal dominance in CD8 properties throughout the response. Aiming to increase clonal dominance, we examined high-frequency transferred P14 T-cell receptor transgenic (TCR Tg) cells. Under these conditions LCMV is cleared faster, and accordingly we found an accelerated response. However, when Tg and endogenous cells were studied in the same mice, where they should be subjected to the same antigen load, they showed overlapping properties, and the presence of P14 cells did not modify endogenous responses to other...

Research paper thumbnail of Optineurin Insufficiency Impairs IRF3 but Not NF- B Activation in Immune Cells

The Journal of Immunology, 2013

Research paper thumbnail of T Cells in G1 Provide a Memory-Like Response to Secondary Stimulation

The Journal of Immunology, 2005

The commitment of naive T cells to proliferate is a function of the strength and duration of stim... more The commitment of naive T cells to proliferate is a function of the strength and duration of stimuli mediated by the TCR and coreceptors. Ranges of 2-20 h of stimulation have been reported as necessary in vitro. Whether T cells actually experience uninterrupted stimulation for such long periods under physiological conditions is controversial. Here we ask whether commitment to proliferate requires continuous stimulation, or can T cells integrate intermittent periods of stimulation. T cells were stimulated for two short-term (subthreshold) periods (5-7 h) either sequentially or separated by an interval of rest. Naive lymph node T cells were able to integrate interrupted stimulation, even when the duration of rest was as long as 2 days. Furthermore, when shortterm-stimulated T cells were separated by density, three populations were observed: low density blasts, intermediate density G 1 cells, and high density G 0 cells. Low density cells progressed to division without further stimulation, whereas G 0 and G 1 cells remained undivided. However, after a period of rest, a second subthreshold stimulation caused the G 1 but not the G 0 fraction to quickly proceed through the cell cycle. We conclude that noncycling T cells in the G 1 phase of the cell cycle remain in a state of readiness for prolonged periods of time, and may represent a population of memory-like effectors capable of responding rapidly to antigenic challenge.

Research paper thumbnail of Regulation of Constitutive TCR Internalization by the  -Chain

The Journal of Immunology, 2002

The ability of a T cell to be activated is critically regulated by the number of TCRs expressed o... more The ability of a T cell to be activated is critically regulated by the number of TCRs expressed on the plasma membrane. Cell surface TCR expression is influenced by dynamic processes such as synthesis and transport of newly assembled receptors, endocytosis of surface TCR, and recycling to the plasma membrane of internalized receptors. In this study, the internalization of fluorescently labeled anti-TCR Abs was used to analyze constitutive endocytosis of TCRs on T cells, and to investigate the role of the zeta-chain in this process. We found that cell surface TCRs lacking zeta were endocytosed more rapidly than completely assembled receptors, and that reexpression of full-length zeta led to a dose-dependent decrease in the rate of TCR internalization. Rapid TCR internalization was also observed with CD4(+)CD8(+) thymocytes from zeta-deficient mice, whereas TCR internalization on thymocytes from CD3-delta deficient animals was slow, similar to that of wild-type thymocytes. This identifies a specific role for zeta in the regulation of constitutive receptor internalization. Furthermore, chimeric zeta molecules containing non-native intracellular amino acid sequences also led to high levels of TCR expression and reduced TCR cycling. These effects were dependent solely on the length of the intracellular tail, ruling out a role for intracellular zeta-specific interactions with other molecules as a mechanism for regulating TCR internalization. Rather, these findings strongly support a model in which the zeta-chain stabilizes TCR residency on the cell surface, and functions to maintain cell surface receptor expression by sterically blocking internalization sequences in other TCR components.

Research paper thumbnail of CD70 Is Downregulated by Interaction with CD27

The Journal of Immunology, 2013

Research paper thumbnail of CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response

Journal of Experimental Medicine, 2007

Research paper thumbnail of The CD8+ memory T-cell state of readiness is actively maintained and reversible

Blood, 2009

The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen e... more The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G(1) to the G(0) cell-cycle state and responded to stimulation like naive T cells, as assessed by proliferation, dependence upon costimulation, and interferon-gamma production, without losing cell surface markers associated with memory. The memory state was maintained by signaling via members of the tumor necrosis factor receptor superfamily, CD27 and 4-1BB. Foxo1, a transcription factor involved in T-cell quiescence, was reduced in memory cells, and stimulation of naive CD8 cells via CD27 caused Foxo1 to be phosphorylated and emigrate from the nucleus in a phosphatidylinositol-3 kinase-dependent manner. Consistent with these results, maintenance of G(1) in vivo was compromised in antigen-specific memory T cells in vesicular stomatitis virus-infected CD27-deficient mice. Therefore, sustaining the functional phenotype of T memory cells requires active signaling and maintenance.

Research paper thumbnail of Dynamic regulation of IL-7 receptor expression is required for normal thymopoiesis

Blood, 2004

IL-7 receptor (IL-7R) levels are tightly controlled during ontogeny: high on DN cells, absent on ... more IL-7 receptor (IL-7R) levels are tightly controlled during ontogeny: high on DN cells, absent on DP cells, and high once again on thymocytes undergoing positive selection. To determine if loss of IL-7-mediated survival signals in DP cells is necessary for normal antigen-specific selection, we created T-lineage specific IL-7R α chain (IL-7Rα) transgenic (Tg) mice in which IL-7R is expressed throughout ontogeny. There was no effect of the IL-7Rα Tg on negative selection. Surprisingly, however, although the thymi of IL-7Rα Tg mice were comparable at birth, there was a decrease in thymocyte number as the mice aged. This was found to be due to competition between DN and IL-7Rexpressing DP cells for endogenous IL-7, which resulted in decreased levels of Bcl-2 in DN cells, increased DN apoptosis, and decreased DN cell number. Therefore, the downregulation of IL-7R on DP cells is an "altruistic" act required for maintaining an adequate supply of local IL-7 for DN cells.

Research paper thumbnail of Epitope Specificity and Relative Clonal Abundance Do Not Affect CD8 Differentiation Patterns during Lymphocytic Choriomeningitis Virus Infection

Journal of Virology, 2009

To evaluate the impact of immunodominance on CD8 T-cell properties, we compared the functional pr... more To evaluate the impact of immunodominance on CD8 T-cell properties, we compared the functional properties of dominant and subdominant populations in the response to lymphocytic choriomeningitis virus (LCMV). To improve functional discrimination, in addition to the usual tests of phenotype and function, we used a sensitive technique that allows the screening of all CD8 effector genes simultaneously in single cells. Surprisingly, these methods failed to reveal a major impact of clonal dominance in CD8 properties throughout the response. Aiming to increase clonal dominance, we examined high-frequency transferred P14 T-cell receptor transgenic (TCR Tg) cells. Under these conditions LCMV is cleared faster, and accordingly we found an accelerated response. However, when Tg and endogenous cells were studied in the same mice, where they should be subjected to the same antigen load, they showed overlapping properties, and the presence of P14 cells did not modify endogenous responses to other LCMV epitopes or a perturbed immunodominance hierarchy in the memory phase. Using allotype-labeled Tg cells, we found that during acute infection up to 80% downregulated their TCR and were undetectable by tetramer binding, and that tetramer-negative and tetramer-positive cells had very different features. Since Tg cells are not available to evaluate immune responses in humans and, in many cases, are not available from the mouse, the tetramer-based evaluation of early immune responses in most situations of high viremia may be incomplete and biased.