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The Journal of Immunology, 2005, 175: 2066-2069.
Copyright © 2005 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: A Single MHC Class Ia Is Sufficient for CD8 Memory T Cell Differentiation 1

Matthew A. Williams and Michael J. Bevan2

Department of Immunology and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Recent studies have suggested a role for MHC class Ib molecules in providing signals for memory T cell differentiation during the early phases of acute infection. To test this hypothesis, we assessed the development of effector and memory CD8 T cells in transgenic mice expressing a single chain H-2Dd/{beta}2-microglobulin ({beta}2M) fusion protein on a {beta}2M-deficient background. These mice thus express a single MHC class Ia in the absence of all other {beta}2M-dependent class Ia and Ib molecules. Following infection with a recombinant vaccinia virus expressing a known Dd-restricted epitope from HIV-1 gp160, the development of effector and memory cells CD8 T cells was comparable to control mice. Furthermore, these memory cells responded rapidly and robustly to antigenic restimulation. Therefore, we conclude that full CD8 memory differentiation requires only a single MHC class Ia chain, ruling out a requirement for MHC class Ib molecules in this process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Following acute infection, naive Ag-specific CD8 T cells undergo a remarkable period of activation and proliferation, with as much as a 10,000- to 50,000-fold expansion in numbers (1). Following the peak of the primary response, 90–95% of the activated effector cells die, leaving behind a population of stable, long-lived memory cells (2, 3). These cells are characterized by their ability to confer long-term protection against reinfection due to their increased precursor frequency, faster response time, and ability to persist virtually indefinitely in the absence of Ag (4).

The early events following acute infection that direct some CD8 T cells to become end-stage effector cells while directing others to become long-lived memory cells remain largely unexplained. A more thorough understanding of these mechanisms will not only shed light on the fundamental immunological question of how long-term protective immunity is established, but also provide insight into the design of safer vaccines that can more effectively generate memory cells in vivo. One recent study identified IL-7R{alpha} as a marker for memory cell precursors (5). A small portion (~10%) of effector CD8 T cells at the peak of the immune response were found to express IL-7R{alpha}, and these cells preferentially survived to become long-lived, functionally protective memory cells. Expression of IL-7R{alpha} was also shown to be functionally important for the survival of these memory precursors.

Recent work has also implicated a role for MHC class Ib molecules in the development of T cell memory (6, 7). One study correlated the transient expression of the CD8{alpha}{alpha} homodimer on Ag-specific CD8 T cells in the spleen with that of IL-7R{alpha}, indicating that early CD8{alpha}{alpha} expression may also be a marker for T cell memory development. Transfer of the CD8{alpha}{alpha}-expressing effector cell subset showed their preferential survival and development into memory cells. Furthermore, memory development was impaired in mice that lacked the ability to up-regulate CD8{alpha}{alpha} on CD8{alpha}{beta} T cells during acute infection, leading the authors to suggest that CD8{alpha}{alpha} binding to its ligand, the MHC class Ib molecule TL, is a necessary step in the differentiation of CD8 memory precursors (7). Because TL binds CD8{alpha}{alpha} homodimers with much greater affinity than CD8{alpha}{beta} heterodimers, and without the need for peptide specificity (8), it has been proposed that this interaction can modify lck signaling by redirecting CD8{alpha}{alpha} away from the TCR activation complex (9).

We sought to test directly whether nonclassical MHC molecules played a role in memory T cell development by studying immune responses to acute infection in transgenic mice that express a single chain (Sc) 3 H-2Dd under the control of a truncated MHC class I promoter in which the N terminus of the H chain is covalently linked to the C terminus of {beta}2M (10, 11). Because the transgenic mice are on a {beta}2-microglobulin ({beta}2M)-deficient background (hereafter referred to as ScDd{beta}2M–/–), no other {beta}2M-dependent MHC class Ia or class Ib molecules, such as TL, is expressed. Following infection with a recombinant vaccinia virus (rVV; Ref. 3) (vPE16; Ref.12) expressing an immunodominant Dd-restricted epitope from HIV-1 gp160 (P18-I10) (13), we observe normal development of both effector and memory CD8 populations. Both effector and memory CD8 T cells in ScDd{beta}2M–/– mice express levels of effector cytokines and IL-7R{alpha} that are comparable to control mice. Furthermore, memory T cells derived in the transgenic mice respond robustly following restimulation, leading us to conclude that a single MHC class Ia is sufficient for the development of CD8 T cell memory, and that MHC class Ib molecules are not required in this process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Disclosures
 References
 
Mice

Six- to 8-wk-old B10.D2 and C57BL/6 mice were purchased from The Jackson Laboratory. C57BL/6NCr tSc{beta}2mDd transgenic mice expressing a Sc H-2Dd/{beta}2M fusion construct were kindly provided by D. Margulies (Molecular Biology Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD) and maintained on a B6.129-B2mtm1Jae ({beta}2M–/–; Taconic Farms) background at our breeding facilities. These mice will hereafter be referred to as ScDd {beta}2M–/–. All mouse experiments were performed with the approval of the Institutional Animal Care and Use Committee at the University of Washington.

Virus infections

Mice 8–12 wk of age were infected with 2 x 106 PFU i.p. of a rVV expressing HIV-1 envelope glycoprotein gp160 (vPE16; kindly provided by B. Moss, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD) (12). The P18-I10 peptide (RGPGRAFVTI) of this protein is H-2Dd-restricted (13).

Intracellular cytokine staining and peptides

Intracellular cytokine staining was performed essentially as described (14). Splenocytes, mesenteric lymph node cells, and liver lymphocytes were resuspended in RP-10 (RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 10 mM HEPES, 0.5 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin). A total of 2 x 106 cells/well were plated in 96-well plates in the presence of 1 µl/ml Brefeldin A (GolgiPlug, Cytofix/Cytoperm kit; BD Pharmingen) with or without the appropriate peptide for 4–5 h at 37°C. P18IIIB-I10 peptide (RGPGRAFVTI) was added at a concentration of 0.1 µg/ml. Following the incubation, cells were stained with CD8-FITC and permeabilized and stained for intracellular cytokine expression using reagents provided in the Cytofix/Cytoperm kit according to the manufacturer’s instructions (BD Pharmingen). The Abs used were IFN-{gamma}-allophycocyanin and IL-2-PE or IL-7R{alpha}-PE (BD Pharmingen), and cells were subsequently analyzed by flow cytometry.

In vitro restimulations

Splenocytes were incubated in 5 µM CFSE in RPMI 1640 (Molecular Probes). After 10 min the staining was halted by the addition of cold RPMI 1640. Cells were washed, resuspended in RP-10 at 2 x 106 cells/well in a 96-well plate, and incubated with 100 nM, 10 nM, or 1 nM P18-I10 peptide. Cells were harvested 3 or 5 days later and analyzed by flow cytometry for CFSE dilution. In duplicate wells, Brefeldin A (GolgiPlug, Cytofix/Cytoperm kit; BD Pharmingen) was added for the final 3 h of stimulation, and cells were subsequently stained and analyzed for intracellular IFN-{gamma} expression.

Ab staining and FACS analysis

Cells were stained with the following Abs: CD8-FITC, CD8-PE, IL-2-PE, IL-7R{alpha}-PE, CD25-allophycocyanin, IFN-{gamma}-allophycocyanin, H-2Dd-FITC, H-2Kb-PE, CD4-PerCP, CD8-allophycocyanin (BD Pharmingen). Cells were analyzed using a FACSCalibur (BD Biosciences) and FlowJo software (Tree Star).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Disclosures
 References
 
ScDd{beta}2M–/– mice, which express a Dd/{beta}2M fusion protein on a {beta}2M-deficient background, have been previously characterized as generating normal numbers of functionally responsive CD8 T cells in the periphery (11). To confirm this, we stained these mice for expression of H2-Kb, H-2Dd, CD4, and CD8 in the spleen and compared them to wild-type mice. As expected, we found that only H-2Dd is detectably expressed in the Dd-transgenic mice, and they generate close to normal numbers of CD8 T cells in the periphery (Fig. 1).



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  		FIGURE 1. ScDd{beta}2M–/– mice express only a single MHC Ia but develop normal numbers of CD8 T cells in the periphery. Splenocytes from wild-type or ScDd{beta}2M–/– mice were stained for the expression of Kb, Dd, CD4, and CD8, as labeled, and analyzed by flow cytometry. Percentages in the lower panels indicate the percentage of CD4+ or CD8+ in the spleen.

 
To test the ability of these mice to respond to acute infection, we infected them with 2 x 106 PFU i.p. with a rVV expressing the HIV-1 envelope glycoprotein gp160 (vPE16). Infection with this virus has previously been shown to stimulate a response to the H-2Dd-restricted P18-I10 peptide of the glycoprotein (11). In accordance with this, we observed a robust response to this epitope in the spleen by day 8 postinfection (p.i.) in both wild-type B10.D2 and ScDd{beta}2M–/– mice, as observed by intracellular IFN-{gamma} staining following ex vivo restimulation (Fig. 2, A and C). In fact, the response to this epitope in the transgenic mice was three to five times larger than that seen in B10.D2 controls. This observation may be due either to a higher precursor frequency of Ag-specific cells, as the entire CD8 repertoire is H-2Dd-restricted, or to a lack of competition with epitopes restricted to other class I MHC. Similar results were obtained in the lymph nodes and liver (data not shown).



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  		FIGURE 2. Mice expressing a single MHC Ia and lacking MHC Ib expression generate normal numbers of effector and memory CD8 T cells following viral infection. B10.D2 mice and ScDd{beta}2M–/– mice 8–12 wk of age were infected with 2 x 106 PFU vPE16 i.v. Splenocytes were harvested at either 8 or 90 days postinfection, incubated with or without 100 nM P18-I10 peptide in the presence of Brefeldin A for 4 h, and stained for the expression of CD8 and IFN-{gamma}. A, Representative flow plots at 8 days postinfection, near the peak of the immune response, are displayed for splenocytes from both groups of mice incubated with either 100 nM peptide or medium alone. B, Representative flow plots at 90 days postinfection, following the establishment of stable memory, are displayed for splenocytes from both groups of mice incubated with either 100 nM peptide or medium alone. Numbers in A and B indicate the percent of whole spleen that are CD8+IFN-{gamma}+. C, Absolute numbers of CD8+IFN-{gamma}+ cells in the spleen at 8 and 90 days postinfection. Percentages represent the percent of the peak (day 8) immune response that remains at day 90. Error bars represent SEM. We analyzed three mice per group at each time point, and the results are representative of two separate experiments.

 
At 90 days p.i. the frequency of P18-I10-specific memory CD8 T cells was assessed in the spleen, lymph nodes, and liver. Memory cells were readily detectable in the spleen in both the B10.D2 controls and the ScDd{beta}2M–/– mice. The numbers of Ag-specific cells corresponded with the extent of the initial primary response, with the B10.D2 memory cells representing 5.2% and the ScDd{beta}2M–/– memory cells representing 12.8% of the initial response (Fig. 2, B and C). Once again, similar results were obtained upon analysis of lymph nodes cells and liver lymphocytes in ScDd{beta}2M–/– mice (data not shown).

Furthermore, the memory cells present in the transgenic mice are similar in phenotype and functional capacity to control mice, as measured by levels of IFN-{gamma} and IL-2 production, and expression of IL-7R{alpha}. In both control B10.D2 and ScDd{beta}2M–/– mice, 80–90% of the P18-I10-specific CD8 memory T cells also expressed IL7R{alpha}, and ~50% of the memory cells coproduced IL-2 along with IFN-{gamma} (Fig. 3). Furthermore, the levels of IFN-{gamma} produced by the memory cells in each group of mice was equivalent, as measured by mean fluorescence intensity (MFI) (data not shown).



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  		FIGURE 3. Memory cells from ScDd{beta}2M–/– mice express normal levels of IL-7R{alpha} and IL-2. At 90 days p.i., splenocytes were assessed for the expression of IFN-{gamma} following a 4-h restimulation in the presence of Brefeldin A. Cells were costained for expression of IL-7R{alpha} (upper panels) or production of IL-2 (lower panels). All flow plots are gated on CD8+ cells. The percentages indicate the percent of IFN-{gamma}+ cells that are IL-7R{alpha}high or IL-2+, respectively. Results are representative of two separate experiments.

 
We sought to determine whether these cells were equally capable of responding to restimulation. Splenocytes from ScDd{beta}2M–/– or B10.D2 memory mice were stained with CFSE and cultured with or without 10 nM P18-I10 peptide for either 3 or 5 days in vitro. Based on the number of cells that diluted their CFSE, as well as the number of divisions, memory cells in both groups of mice proliferated comparably in response to restimulation, resulting in a 3- to 5-fold expansion by day 3 and a 20- to 30-fold expansion by day 5 for both groups (Fig. 4, A and B). Furthermore, memory cells from both groups of mice generated comparable and robust IFN-{gamma} responses (Fig. 4C). Responses for each group were comparable over a range of peptide concentrations from 1 nM to 1 µM (data not shown).



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  		FIGURE 4. Memory cells from ScDd{beta}2M–/– mice generate unimpaired secondary responses upon rechallenge. Splenocytes from B10.D2 or Dd/{beta}2M x {beta}2M–/– memory mice (day 90 p.i.) were labeled with CFSE and cultured in the presence of 10 nM P18-I10 peptide in vitro for either 3 or 5 days. A, Representative flow plots display CFSE division of CD8+ cells after 5 days of culture in vitro with P18-I10 peptide or medium alone. B, The number of divided cells, as assessed by CFSE dilution, was calculated at days 3 and 5 following restimulation. To calculate fold expansion, the number of CFSE-diluted cells in the unstimulated well was subtracted from the number of CFSE-diluted cells in the peptide-stimulated well, then divided by the starting number of Ag-specific memory cells. Three mice per group were analyzed, and the error bars represent the SEM. C, After 5 days in vitro restimualtion, Brefeldin A was added to some wells for 3 h, and the cells were subsequently stained for expression of IFN-{gamma}. Representative flow plots, gated on CD8+ cells, display the frequency of IFN-{gamma}+ and IFN-{gamma}– CFSE-diluted cells for each group, as labeled.

 
These experiments demonstrate that a single MHC class Ia, in the absence of any other {beta}2M-dependent MHC class Ia or Ib, can dictate the full differentiation of CD8 memory cells. Effector and memory cells generated in ScDd{beta}2M–/– mice are phenotypically and functionally similar to those seen in B10.D2 control mice. Furthermore, the resulting memory cells retain the capacity to respond rapidly and robustly to restimulation. Therefore, it is clear that TL, along with other {beta}2M-dependent class Ib molecules, does not play an obligatory role in the differentiation of CD8 memory following acute infection. Nevertheless, the possibility exists that individual class Ib molecules may play positive or negative roles in providing signals for T cell differentiation that are not readily apparent in our mice which lack all {beta}2M-dependent class Ib molecules. It also remains possible that CD8{alpha}{alpha} has an alternative ligand, and that binding to this ligand relays an important memory differentiation signal.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Howard Hughes Medical Institute and National Institutes of Health Grant AI19335 (to M.J.B.) and by Ruth L. Kirchstein National Research Service Award Fellowship AI056809 (to M.A.W.). Back

2 Address correspondence and reprint requests to Dr. Michael J. Bevan, Department of Immunology and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195. E-mail address: mbevan{at}u.washington.edu Back

3 Abbreviations used in this paper: Sc, single chain; {beta}2M, {beta}2-microglobulin; rVV, recombinant vaccinia virus; MFI, mean fluorescence intensity. Back

Received for publication May 9, 2005. Accepted for publication June 9, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 

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