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T helper subset differentiation in the absence of invariant chain.

Brown DR, Swier K, Moskowitz NH, Naujokas MF, Locksley RM, Reiner SL - J. Exp. Med. (1997)

Bottom Line: We wished to determine whether this unexpected efficiency of T helper subset induction despite inefficient T cell stimulation could be modeled in vitro.In the presence of rIL-12 or rIL-4 naive parasite-specific transgenic T cells could mature into IFN-gamma-or IL-4-secreting T helper cells, respectively, even when antigen presentation was suboptimal or antigen dose was submitogenic.These experiments demonstrate that activation of T helper cells to a threshold required for IL-2 production or proliferation is not required to achieve induction of disease-regulating T helper cell effector functions, and that pathogen-associated secondary activation signals may facilitate the full differentiation of T helper subsets during limiting presentation of antigenic peptides.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Gwen Knapp Center for Lupus & Immunology Research, University of Chicago, Illinois 60637, USA.

ABSTRACT
The outcome of murine infection with Leishmania major is regulated by major histocompatibility complex class II-restricted T helper cells. Invariant chain-deficient (Ii -/-) mice have impaired ability to present major histocompatibility complex class II-restricted antigens, and reduced numbers of CD4+ T cells. Despite these deficits, C57BL/6 Ii -/- mice controlled L. major infection comparably to wild-type mice. As assessed by mRNA analysis and in vitro antigen restimulation for IFN-gamma, Ii -/- mice had normal induction of Th1 subset differentiation even though antigen-dependent proliferation of their lymph node cells was substantially compromised. In addition, BALB/c Ii -/- mice exhibited a progressive course of infection and Th2 effector cell development that were comparable to that seen in wild-type BALB/c mice. We wished to determine whether this unexpected efficiency of T helper subset induction despite inefficient T cell stimulation could be modeled in vitro. In the presence of rIL-12 or rIL-4 naive parasite-specific transgenic T cells could mature into IFN-gamma-or IL-4-secreting T helper cells, respectively, even when antigen presentation was suboptimal or antigen dose was submitogenic. These experiments demonstrate that activation of T helper cells to a threshold required for IL-2 production or proliferation is not required to achieve induction of disease-regulating T helper cell effector functions, and that pathogen-associated secondary activation signals may facilitate the full differentiation of T helper subsets during limiting presentation of antigenic peptides.

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Ii −/− mice develop type 1 immune responses  during infection with L. major.  (A) Lymph node cells from the  designated mice were collected 8  wk after infection with L. major,  and incubated as described in the  Materials and Methods, with or  without soluble Leishmania antigens for 48 h. Supernatants were  assayed for IFN-γ and IL-4 by  ELISA. Points indicate quantities  present in antigen-stimulated  wells. All incubations in the absence of antigen resulted in cytokine recoveries <7 U/ml, except for one β2m −/− animal with an IFN-γ level of 19 U/ml, and the  BALB/c with an IL-4 level of 100 U/ml. Data points represent means  and standard deviations of triplicate determinations, and are representative of five separate experiments. (B) Total RNA from lymph node cells  from infected Ii +/+ (+/+), −/− (−/−), or BALB/c (B/c) mice was  reverse transcribed and subjected to PCR amplification in the presence of  a competitor construct containing mutated cDNAs (upper bands) ∼75 bp  larger than the authentic transcripts (lower bands). After standardizing the  input cDNA for the amounts of the constitutively expressed HPRT gene  product, the amounts of IFN-γ and IL-4 were determined using the requisite primers. These results are representative of three separate experiments.
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Figure 3: Ii −/− mice develop type 1 immune responses during infection with L. major. (A) Lymph node cells from the designated mice were collected 8 wk after infection with L. major, and incubated as described in the Materials and Methods, with or without soluble Leishmania antigens for 48 h. Supernatants were assayed for IFN-γ and IL-4 by ELISA. Points indicate quantities present in antigen-stimulated wells. All incubations in the absence of antigen resulted in cytokine recoveries <7 U/ml, except for one β2m −/− animal with an IFN-γ level of 19 U/ml, and the BALB/c with an IL-4 level of 100 U/ml. Data points represent means and standard deviations of triplicate determinations, and are representative of five separate experiments. (B) Total RNA from lymph node cells from infected Ii +/+ (+/+), −/− (−/−), or BALB/c (B/c) mice was reverse transcribed and subjected to PCR amplification in the presence of a competitor construct containing mutated cDNAs (upper bands) ∼75 bp larger than the authentic transcripts (lower bands). After standardizing the input cDNA for the amounts of the constitutively expressed HPRT gene product, the amounts of IFN-γ and IL-4 were determined using the requisite primers. These results are representative of three separate experiments.

Mentions: Popliteal lymph node cells draining the lesions of infected mice were restimulated in vitro with soluble parasite antigens to assess the qualitative cytokine response. IFN-γ was recovered from supernatants of stimulated cells from infected Ii −/− and β2m −/− Ii −/− mice at levels comparable to or greater than cells from Ii +/− mice (Fig. 3 A). Production of IFN-γ in vitro was reduced to less-than-detectable levels if anti–class II monoclonal antibody, but not an isotype control antibody, was included during the period of antigen stimulation (data not shown). IL-4, which was readily recovered by stimulation of cells from susceptible BALB/c mice, was not detected. Similar results were obtained analyzing the expression of IFN-γ and IL-4 transcripts in lymph node cells immediately after removal from infected animals (Fig. 3 B). Taken together, these data suggest that the deficiency in Ii expression does not impede the development of a class II– dependent type 1 immune response to L. major. Th1 effector development was comparable in Ii −/− mice on H-2b, H-2k, and H-2d backgrounds, despite the differences in efficient assembly of these different class II molecules in the absence of Ii (27).


T helper subset differentiation in the absence of invariant chain.

Brown DR, Swier K, Moskowitz NH, Naujokas MF, Locksley RM, Reiner SL - J. Exp. Med. (1997)

Ii −/− mice develop type 1 immune responses  during infection with L. major.  (A) Lymph node cells from the  designated mice were collected 8  wk after infection with L. major,  and incubated as described in the  Materials and Methods, with or  without soluble Leishmania antigens for 48 h. Supernatants were  assayed for IFN-γ and IL-4 by  ELISA. Points indicate quantities  present in antigen-stimulated  wells. All incubations in the absence of antigen resulted in cytokine recoveries <7 U/ml, except for one β2m −/− animal with an IFN-γ level of 19 U/ml, and the  BALB/c with an IL-4 level of 100 U/ml. Data points represent means  and standard deviations of triplicate determinations, and are representative of five separate experiments. (B) Total RNA from lymph node cells  from infected Ii +/+ (+/+), −/− (−/−), or BALB/c (B/c) mice was  reverse transcribed and subjected to PCR amplification in the presence of  a competitor construct containing mutated cDNAs (upper bands) ∼75 bp  larger than the authentic transcripts (lower bands). After standardizing the  input cDNA for the amounts of the constitutively expressed HPRT gene  product, the amounts of IFN-γ and IL-4 were determined using the requisite primers. These results are representative of three separate experiments.
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Related In: Results  -  Collection

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Figure 3: Ii −/− mice develop type 1 immune responses during infection with L. major. (A) Lymph node cells from the designated mice were collected 8 wk after infection with L. major, and incubated as described in the Materials and Methods, with or without soluble Leishmania antigens for 48 h. Supernatants were assayed for IFN-γ and IL-4 by ELISA. Points indicate quantities present in antigen-stimulated wells. All incubations in the absence of antigen resulted in cytokine recoveries <7 U/ml, except for one β2m −/− animal with an IFN-γ level of 19 U/ml, and the BALB/c with an IL-4 level of 100 U/ml. Data points represent means and standard deviations of triplicate determinations, and are representative of five separate experiments. (B) Total RNA from lymph node cells from infected Ii +/+ (+/+), −/− (−/−), or BALB/c (B/c) mice was reverse transcribed and subjected to PCR amplification in the presence of a competitor construct containing mutated cDNAs (upper bands) ∼75 bp larger than the authentic transcripts (lower bands). After standardizing the input cDNA for the amounts of the constitutively expressed HPRT gene product, the amounts of IFN-γ and IL-4 were determined using the requisite primers. These results are representative of three separate experiments.
Mentions: Popliteal lymph node cells draining the lesions of infected mice were restimulated in vitro with soluble parasite antigens to assess the qualitative cytokine response. IFN-γ was recovered from supernatants of stimulated cells from infected Ii −/− and β2m −/− Ii −/− mice at levels comparable to or greater than cells from Ii +/− mice (Fig. 3 A). Production of IFN-γ in vitro was reduced to less-than-detectable levels if anti–class II monoclonal antibody, but not an isotype control antibody, was included during the period of antigen stimulation (data not shown). IL-4, which was readily recovered by stimulation of cells from susceptible BALB/c mice, was not detected. Similar results were obtained analyzing the expression of IFN-γ and IL-4 transcripts in lymph node cells immediately after removal from infected animals (Fig. 3 B). Taken together, these data suggest that the deficiency in Ii expression does not impede the development of a class II– dependent type 1 immune response to L. major. Th1 effector development was comparable in Ii −/− mice on H-2b, H-2k, and H-2d backgrounds, despite the differences in efficient assembly of these different class II molecules in the absence of Ii (27).

Bottom Line: We wished to determine whether this unexpected efficiency of T helper subset induction despite inefficient T cell stimulation could be modeled in vitro.In the presence of rIL-12 or rIL-4 naive parasite-specific transgenic T cells could mature into IFN-gamma-or IL-4-secreting T helper cells, respectively, even when antigen presentation was suboptimal or antigen dose was submitogenic.These experiments demonstrate that activation of T helper cells to a threshold required for IL-2 production or proliferation is not required to achieve induction of disease-regulating T helper cell effector functions, and that pathogen-associated secondary activation signals may facilitate the full differentiation of T helper subsets during limiting presentation of antigenic peptides.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Gwen Knapp Center for Lupus & Immunology Research, University of Chicago, Illinois 60637, USA.

ABSTRACT
The outcome of murine infection with Leishmania major is regulated by major histocompatibility complex class II-restricted T helper cells. Invariant chain-deficient (Ii -/-) mice have impaired ability to present major histocompatibility complex class II-restricted antigens, and reduced numbers of CD4+ T cells. Despite these deficits, C57BL/6 Ii -/- mice controlled L. major infection comparably to wild-type mice. As assessed by mRNA analysis and in vitro antigen restimulation for IFN-gamma, Ii -/- mice had normal induction of Th1 subset differentiation even though antigen-dependent proliferation of their lymph node cells was substantially compromised. In addition, BALB/c Ii -/- mice exhibited a progressive course of infection and Th2 effector cell development that were comparable to that seen in wild-type BALB/c mice. We wished to determine whether this unexpected efficiency of T helper subset induction despite inefficient T cell stimulation could be modeled in vitro. In the presence of rIL-12 or rIL-4 naive parasite-specific transgenic T cells could mature into IFN-gamma-or IL-4-secreting T helper cells, respectively, even when antigen presentation was suboptimal or antigen dose was submitogenic. These experiments demonstrate that activation of T helper cells to a threshold required for IL-2 production or proliferation is not required to achieve induction of disease-regulating T helper cell effector functions, and that pathogen-associated secondary activation signals may facilitate the full differentiation of T helper subsets during limiting presentation of antigenic peptides.

Show MeSH
Related in: MedlinePlus