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The Journal of Immunology, 2005, 175: 6615-6623.
Copyright © 2005 by The American Association of Immunologists

Tissue-Resident Memory CD8+ T Cells Can Be Deleted by Soluble, but Not Cross-Presented Antigen1

Cheng-Hong Wei*, Rebecca Trenney*, Manuel Sanchez-Alavez{dagger}, Kristi Marquardt*, David L. Woodland{ddagger}, Steven J. Henriksen{dagger} and Linda A. Sherman2,*

* Department of Immunology, and {dagger} Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037; and {ddagger} Trudeau Institute, Saranac Lake, NY 12983


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Under noninflammatory conditions, both naive and central memory CD8 T cells can be eliminated in the periphery with either soluble peptide or cross-presented Ag. Here, we assess the tolerance susceptibility of tissue-resident memory CD8 T cells in mice to these two forms of tolerogen. Soluble peptide specifically eliminated the majority of memory CD8 cells present in both lymphoid and extralymphoid tissues including lung and liver, but was unable to reduce the number present in the CNS. In contrast, systemic cross-presentation of Ag by dendritic cells resulted in successful elimination of memory cells only from the spleen, with no significant reduction in the numbers of tissue-resident memory cells in the lung. The fact that tissue-resident memory cells were unable to access cross-presented Ag suggests that either the memory cells in the lung do not freely circulate out of the tissue, or that they circulate through a region in the spleen devoid of cross-presented Ag. Thus, although tissue-resident memory cells are highly susceptible to tolerance induction, both the form of tolerogen and location of the T cells can determine their accessibility to tolerogen and the degree to which they are successfully deleted from specific tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Memory T lymphocytes play an important role in the host’s defense to recurrent infections (1). However, under certain circumstances, their presence poses a major barrier to disease resolution. In chronic autoimmune diseases with multiple relapses, autoreactive memory T cells can be the major source of tissue-destructive cells and thus a major obstacle in resolution of disease (2, 3). Also, there is evidence that a large proportion of allospecific T cells are memory T cells, which constitute an obstacle for transplantation tolerance (4, 5, 6, 7, 8). Therefore, efficient tolerance induction of Ag-specific memory T cells has significant potential in autoimmune disease and allograft transplantation.

Several different methods of Ag delivery have proven successful in the tolerance induction of naive CD8 cells in vivo. Oral, intranasal, or systemic delivery of soluble peptide has been used successfully in many studies to tolerize naive CD4 and CD8 T cells (9, 10, 11, 12, 13, 14). In addition, recent advances in our understanding of normal mechanisms of peripheral tolerance have resulted in the development of tolerance protocols that rely on the capacity of dendritic cells (DCs)3 to acquire, process, and cross-present Ag in a tolerogenic form (15, 16, 17). Peripheral deletion of CD8 cells normally occurs when naive cells encounter Ag on quiescent DCs in lymphoid tissue. Exogenous Ag can be acquired by DCs either through endocytosis of dying cells or when they are in close contact with live cells containing Ag (18, 19, 20, 21). These observations have led to the development of techniques that promote tolerance through delivery of Ag to quiescent DCs. For example, spleen cells can be loaded with protein Ag in vitro and forced to undergo apoptosis by incubation under hypotonic conditions. Upon injection in vivo, the dying cells are taken up by DCs that cross-present the Ag to cognate T cells, thereby resulting in deletion of the Ag-specific T cell (17). As an alternative strategy, Ag can be specifically targeted to DCs for cross-presentation by using a mAb specific for the DC surface molecule, DEC-205 (15, 16).

Our laboratory demonstrated recently that either soluble peptide or Ag cross-presented by DCs is highly effective in eliminating both naive and memory T cells from secondary lymphoid tissue (22). However, memory T cells can also reside in parenchymal tissues, and these represent an important component of the memory T cell pool. A number of studies have demonstrated that following clearance of pathogen by an immune response, large numbers of Ag-specific CD8+ T cells persist for long periods of time in peripheral tissues including the lung airways and parenchyma, gut, kidney, liver, and fat pads (23, 24, 25, 26).

In this report, we have assessed the tolerance susceptibility of such tissue-resident memory CD8 T cells specific for influenza virus. Specifically, we have compared two different forms of tolerogen, soluble peptide or Ag cross-presented by DCs, for their ability to eliminate memory cells present in either secondary lymphoid tissue or parenchymal tissue. The results indicate differential susceptibility of tissue-resident memory cells to elimination by these two forms of tolerogen.


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

Female BALB/c mice were purchased from The Jackson Laboratory or the Animal Breeding Facility at The Scripps Research Institute and housed under specific pathogen-free conditions. Female {beta}2-microglobulin-deficient mice ({beta}2M KO) were purchased from The Jackson Laboratory and maintained under sterile conditions at the Animal Breeding Facility at The Scripps Research Institute. Experimental procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Virus and infection

Influenza virus A/PR/8/34 H1N1 (PR8) was grown in the allantoic cavity of 10- to 11-day-old hen’s eggs. Upon isolation, the allantoic fluid was titrated for hemagglutinin (HA) activity using chicken RBC and stored at –70°C. Mice (10–16 wk; >20 g) were anesthetized by i.p. injection with avertin (2,2,2-tribromoethanol). A stock solution was prepared by dissolving 10 g of avertin in 10 ml of amyl alcohol and stored refrigerated. A working solution was prepared by diluting the stock solution 20x in PBS, from which 200 µl was injected per mouse for anesthesia. Mice were infected intranasally with 1 HA U of PR8 virus. Mice were considered to be "memory mice" when they had been infected with influenza a minimum of 40 days previously. Recombinant vaccinia virus expressing the nucleoprotein (NP) gene or the HA gene of influenza virus was kindly provided by J. Yewdell and J. Bennick from the National Institutes of Health (Bethesda, MD). Mice were infected with 106 PFU of recombinant vaccinia virus through i.v. injection.

Peptides

Peptides used in these studies included the following: the Kd-restricted NP147–155 (TYQRTRALV) epitope, the KdHA518–526 (IYSTVASSL) epitope, and ILA-KdHA (ILAIYSTVASSL; hereafter referred to as ILA), which extends the sequence of the nominal peptide by including 3 amino proximal residues in the protein. All peptides were synthesized at The Peptide Synthesis Core Facility at The Scripps Research Institute. Peptide purity was evaluated using reverse-phase HPLC analysis. The KdNP peptide was dissolved in HBSS and 100 µg in 200 µl was injected i.v. KdHA peptide was initially dissolved in DMSO at 20 mg/ml, and then diluted in HBSS, and 100 µg was injected in 200 µl i.v.

Loading of {beta}2M KO spleen cells with ILA peptide

ILA-Ag-loaded spleen cells were prepared by osmotic shock using the method described by Steinman and coworkers (17). Briefly, 150 x 106 splenocytes from {beta}2M KO mice were washed twice in RPMI 1640 and resuspended in 1 ml of hypertonic medium (0.5 M sucrose, 10% w/v polyethylene glycol 1000, and 10 mM HEPES in RPMI 1640 (pH 7.2)) containing 0.05 mg/ml ILA peptide, for 10 min at 37°C. Thirteen milliliters of prewarmed hypotonic medium (40% H2O, 60% RPMI 1640) was added, and the cells were incubated for an additional 2 min at 37°C. Immediately thereafter, the cells were pelleted by centrifugation, washed twice with ice-cold HBSS, and 30 x 106 in 0.2 ml of ILA-loaded splenocytes were injected i.v. into each recipient mouse as a source of dying cells.

Perfusion and tissue preparation

Mice were anesthetized by i.p. injection with avertin as described above. The thoracic cavity was opened to expose the lungs and heart. Perfusion was performed twice through the right ventricle with 30 ml of HBSS, followed by similar perfusion through the left ventricle. Lungs, liver, brain, and spinal cord were removed and placed in cold complete RPMI 1640 medium (RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS (Gemini Bio-Products), 2 mM glutamine (Invitrogen Life Technologies), 5 x 10–5 M 2-ME (Sigma-Aldrich), and 50 mg/ml gentamicin (Gemini Bio-Products)). Tissues were forced through wire mesh and incubated at 37°C for 60 min in 150 U of collagenase (Invitrogen Life Technologies) per milliliter of complete RPMI 1640 medium (without 2-ME). Cell suspensions were then washed, resuspended in 20 ml of RPMI 1640 (without FCS), and mixed with 10 ml of Percoll. Tissue-resident lymphocytes were isolated from a continuous Percoll (Pharmacia) gradient after centrifugation for 30 min at 4°C at 27,000 xg. Spleen and peripheral lymph nodes (the cervical, auxiliary, brachial, inguinal lymph nodes) were also removed, and peripheral lymph nodes were pooled. Single-cell suspensions were obtained from spleens and lymph nodes by passage through an iron screen and nytex mesh.

Intracellular cytokine staining

To assess the ex vivo production of IFN-{gamma} in response to peptide Ag, single-cell suspensions of purified lymphocytes from various tissues were prepared as described above, and cells were incubated in complete RPMI 1640 medium with 1 µg/ml KdNP or KdHA peptide and 1 µl/ml GolgiPlug solution (BD Pharmingen) for 6 h at 37°C. Cells were then incubated in 100 µl of 2.4G2 hybridoma supernatant (American Type Culture Collection) for 10 min at 4°C to block FcRs. PE-conjugated anti-CD8 was then added, and cells were incubated for an additional 30 min at 4°C. After two washes, intracellular IFN-{gamma} staining was performed according to the manufacturer’s instructions using the Cytofix/Cytoperm Plus kit (BD Pharmingen) and allophycocyanin-conjugated rat anti-mouse IFN-{gamma}. Cells were analyzed on a BD Biosciences FACSort flow cytometer, and data were analyzed using CellQuest software. All Abs for flow cytometry were purchased from BD Pharmingen. The total number of NP- or HA-specific CD8 memory T cells in each tissue was calculated based on the percentage of IFN-{gamma}+ CD8 cells within the total lymphocytes and the average number of viable lymphocytes harvested from each tissue. The percentage of depletion was calculated through the following formula: (number of Ag-specific T cells in control group – number of Ag-specific T cells in tolerance group)/number of Ag-specific T cells in control group x 100%.

Tetramers and tetramer staining

Allophycocyanin-labeled tetramer of H-2Kd complexed with NP peptide was synthesized in the Molecular Biology Core Facility at the Trudeau Institute. Staining with allophycocyanin-Kd-NP tetramer reagents was performed by incubation for 1 h at 4°C, followed by incubation with anti-CD8 PE (BD Pharmingen) on ice for 20 min together with FITC-conjugated Abs specific for CD4, B220, CD11c (BD Pharmingen), which allowed removal of non-CD8 cells during FACS analysis. Stained samples were run on FACSCalibur flow cytometer, and data were analyzed using CellQuest software (BD Biosciences). The percentage of tetramer+ cells among total CD8+ cells was calculated by dividing the number of tetramer+/CD8+ events by the total number of CD8+ events in the live cell gate.

Intracerebroventricular (i.c.v.) injection of soluble peptide

For the i.c.v. injections, mice were equipped with an i.c.v. cannula aimed above the lateral ventricle under anesthesia. As the injection procedure itself produces no pain, the animals are gently held and awake during the injection. For an acute injection, a 30-gauge stainless-steel cannula with 60 cm of tygon microbore tubing (0.03-inch outer diameter, 0.01-inch inner diameter) attached is inserted to the lateral ventricle (bregma, –0.34 mm; lateral, 1.0 mm; ventral, 2.0–2.5 mm). One microliter of soluble KdNP (20 mg/ml) is injected using a pump (rate of injection is 0.2 µl/min; time of injection: 5 min) into each side of the lateral ventricle, so a total amount of 40 µg of NP peptide was injected into both lateral ventricles. Volume is also measured by marks on the injector tubing previously calibrated with a 10-µl Hamilton syringe.

Immunohistology

Spleens were immersed in HBSS and snap-frozen in liquid nitrogen, and 5 µm-thick cryostat sections were cut and fixed in 1% paraformaldehyde for 10 min. These sections were incubated with the primary Abs (rat anti-mouse CD8, CD4, B220, respectively) for 60 min followed by incubation with biotinylated secondary Ab (mouse anti-rat F(ab')2) for 60 min and streptavidin-HRP for 45 min. HRP was detected by a color reaction by incubating with chromagen AEC (Vector) in the dark for 15 min. Slides were counterstained with Mayer’s hematoxylin (Sigma-Aldrich).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Peripheral tolerance of NP-specific memory CD8 cells by soluble NP peptide injection

Seventy days after intranasal infection of BALB/c with the A/PR8(H1N1) strain of influenza virus (PR8), lymphocytes were isolated from lymphoid and nonlymphoid tissues and assessed for the presence of PR8-specific CD8 memory cells based on their ability to produce IFN-{gamma} in response to a brief period of incubation with the Kd-restricted peptide from the viral NP (Fig. 1A). NP-specific CD8 T cells were present in all tissues examined, including peripheral lymph nodes, spleen, lung, liver, and CNS (brain and spinal cord). No NP-specific IFN-{gamma}+ CD8 T cells were found in the tissues of naive uninfected animals (data not shown). As a percentage of CD8 T cells, NP-specific cells were most prominent in the CNS where 20.9% of the CD8 cells produced IFN-{gamma}. The lung also contained a relatively high percentage (1.67%) of NP-specific CD8 cells. However, when the total number of NP-specific CD8 T cells was calculated for each tissue, the largest number was found to be present in the spleen (~120,000), whereas the numbers of cells recovered from the lung and peripheral lymph nodes was much lower (~14,000 and ~8,000, respectively). The CNS and liver yielded small numbers of NP-specific CD8 memory T cells (~4,000 and ~2,000, respectively) (Fig. 1C). These results are in general agreement with previous reports on the distribution of tissue-resident memory CD8 T cells (23).



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  		FIGURE 1. Soluble peptide can tolerize memory T cells in lymphoid and nonlymphoid tissues with the exception of the CNS. Forty days following infection with PR8, mice (three mice per group) received either 100 µg of NP peptide (PR8 + NP peptide) injected i.v. or HBSS (PR8). Thirty days later, both groups were sacrificed, animals were perfused, and lymphocytes were isolated from the indicated tissue. A, NP-specific CD8 cells were detected based on intracellular production of IFN-{gamma}. The percentage of NP-specific T cells among the total CD8+ T cells in each tissue from one representative experiment of three is shown. B, The percentage of HA-specific CD8 T cells in both the control and NP peptide group were assessed by intracellular staining for IFN-{gamma} and FACS analysis. Results represent one experiment of three with similar results. C, The absolute number of NP-specific CD8 memory T cells in each tissue was calculated and plotted. D, The percentage of depletion in each of the indicated tissues was calculated as described in Materials and Methods. The results shown are means and SD of three independent experiments.

 
To determine whether tissue-resident, NP-specific memory CD8 T cells could be eliminated with soluble peptide Ag, mice containing influenza-specific memory cells received a single dose of 100 µg of NP peptide and then rested for a period of 1 mo to allow any potentially anergized T cells to recover responsiveness to secondary stimulation (27), after which time the mice were sacrificed and NP-specific memory CD8 T cells were assessed by intracellular staining for IFN-{gamma}. As shown in Fig. 1, C and D, both the percentage and numbers of NP-specific memory CD8 T cells were substantially decreased in mice treated with NP peptide. With the exception of the CNS, all tissues tested (peripheral lymph nodes, spleen, lung, liver) exhibited a significant reduction in the number of NP-responsive memory cells. Such deletion was Ag specific because the percentage and number of CD8 cells specific for the subdominant epitope from the viral HA were not decreased following the injection of NP peptide (Fig. 1B).

Previously, it has been reported that treatment of lymphocytic choriomeningitis virus-primed mice with a peptide from the viral glycoprotein (gp33) induced severe immunopathology in the spleen, thus resulting in nonspecific suppression of unrelated CTL responses in vivo (28). This was attributed to cytolytic activity by memory T cells against peptide-coated lymphoid cells. However, following injection of soluble NP peptide, we saw no reduction in the number of HA-specific memory cells (Fig. 1B). To further assess the possibility that memory cells may damage peptide-coated tissue, we performed a histological examination of the spleen of peptide-treated mice yet found no obvious pathological changes or differences observed between the spleens isolated from the two groups of mice (data not shown).

The reduction in the number of NP-specific memory CD8 T cells after injection of NP peptide was 60% in peripheral lymph nodes, and >80% of memory CD8 T cells from spleen, lung, and liver (Fig. 1C). Thus, the majority of both central and tissue-resident memory cells were deleted by soluble peptide. Notably, peptide injection did not decrease the number of NP-specific memory T cells in the CNS, possibly because the peptide was unable to pass through the blood-brain barrier to access cells localized within the CNS. However, further attempts to tolerize memory cells localized in the CNS by introducing Ag via i.c.v. injection to lateral ventricles on both sides of the brain, a procedure that would allow peptide to diffuse throughout the CNS, did not significantly reduce the numbers of cells (Fig. 2A). In addition, the combination of systemic delivery (i.v.) and i.c.v. injection of the soluble peptide Ag also failed to eliminate memory cells from the CNS (Fig. 2B). This suggests that memory T cells residing in the CNS do not freely circulate to the periphery; otherwise they would have been tolerized by peptide in the periphery and there would be a decline in the number of NP-specific T cells in the CNS.



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  		FIGURE 2. Intracerebroventricular injection of soluble NP peptide does not tolerize memory CD8 T cells in the CNS. A, Forty days following infection with PR8, i.c.v. injection of 40 µg of NP peptide to both side of the lateral ventricles was performed in one group of the mice (PR8 + i.c.v. NP peptide group) to allow the peptide to diffuse throughout the CNS. The control group of PR8-infected memory mice just received i.c.v. injection of HBSS (PR8 group). Thirty days later, the percentage of NP-specific memory T cells in the CNS of the mice that have received the i.c.v. injection of NP peptide was evaluated by intracellular staining for IFN-{gamma} as detected by FACS, and compared with the control mice. B, Also, the number of NP-specific memory T cells in the CNS of the PR8-infected memory mice that received both i.c.v. and i.v. injections of NP peptide (PR8 + i.c.v & i.v. NP peptide group) was evaluated and compared with the results from control infected mice.

 
Tolerance induction occurred through deletion of specific CTLs

The assay used to detect the presence of memory T cells relied on production of IFN-{gamma} and would not detect cells that may be present yet could not produce IFN-{gamma}, perhaps as the result of exposure to peptide in vivo. To determine whether such nonresponsive memory cells were present, we used H-2KdNP tetramers to detect all NP-specific CD8 cells isolated from the spleens and lungs of peptide-treated and untreated control mice. As shown in Fig. 3, following NP peptide treatment, there was also a significant decline in the number of tetramer-binding CD8 T cells in both the spleen and the lung, and the percentage of depletion as measured by intracellular staining (Fig. 1D) and by tetramer staining (Fig. 3C) was comparable in both tissues, confirming that the majority of NP-specific memory CD8 T cells were actually deleted. Therefore, soluble peptide treatment successfully deleted the majority of memory T cells residing in secondary lymphoid and nonlymphoid tissues.



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  		FIGURE 3. Tolerance of memory CD8 T cells occurs through elimination of peptide-specific CD8 cells. Lymphocytes isolated from spleen (A) and lungs (B) of the control and NP peptide-treated group of mice (three mice/group) from Fig. 1A were incubated with KdNP tetramer and anti-CD8 mAb to detect NP-specific CD8 cells. The background staining in naive spleen and lung were determined to be 0.2 and 0.3%, respectively. C, The mean and SD of the percent depletion of KdNP tetramer binding CD8 T cells by soluble NP peptide. Data represent the results from three independent experiments, each performed using three mice per group.

 
Cross-presented Ag can delete central memory cells yet does not reduce the numbers of tissue-resident memory cells

Peripheral tolerance of both naive and memory cells generally involves T cell recognition of Ags cross-presented by quiescent DCs. To assess the ability of cross-presenting DCs to tolerize tissue-resident memory T cells, we adapted a protocol that was shown previously to lead to efficient deletion of naive CD8 cells (17). Splenocytes were incubated with Ag under hyperosmotic conditions, which results in uptake of Ag and promotes apoptotic death of the cells. Upon injection into mice, the dying cells are taken up by splenic DCs that efficiently process and cross-present the Ag to naive T cells. It has been shown that recognition of cognate Ag cross-presented by DCs in this manner results in abortive activation and deletion of the cells (17). To avoid the need to produce large quantities of viral protein, we modified this protocol by loading spleen cells in vitro with an extended form of the antigenic HA peptide that contained three additional N-terminal residues that are found in the natural protein sequence (ILA). The efficiency of cross-presentation of this longer form of epitope was much higher than that of the original 9-mer peptide (C.H.W. and L.A.S. unpublished results), consistent with the observation of Norbury et al. (29) as well as Shen and Rock (30) that nominal peptide is inefficient for cross-presentation. When splenocytes from {beta}2M KO mice were loaded with the ILA-HA peptide and injected into mice, the Ag was efficiently cross-presented in vivo, as assessed by proliferation of HA specific TCR transgenic cells from clone 4 mice (C.-H. Wei and L. A. Sherman, unpublished results).

To determine whether cross-presented Ag could result in tolerance and deletion of HA-specific memory cells, mice previously infected with influenza virus received ILA-HA-loaded {beta}2M KO spleen cells. As shown in Fig. 4A, the number of HA-specific memory T cells localized in the spleen was reduced as efficiently as in mice that received soluble peptide treatment. In contrast, this form of tolerogen did not significantly reduce the numbers of tissue-resident memory cells in the lungs.



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  		FIGURE 4. Cross-presented Ag tolerizes memory cells residing in the spleen, but not those in the lungs. Forty days following infection with PR8, groups of mice (three mice per group) received either 100 µg of HA peptide injected i.v. or 30 x 106 BALB/c {beta}2M KO spleen cells that had been loaded with the 12-mer ILA peptide through osmotic shock, or HBSS (control). Thirty days later, all groups were sacrificed, and lymphocytes were isolated from spleen and lung. The percentage of IFN-{gamma}+ CD8 T cells was determined by flow cytometry. A, The number of HA-specific T cells present in tissues from each group is shown as mean and SD of three independent experiments. B, The data in A were used to calculate the mean and SD of the percent depletion. Results from one experiment in which a group of three mice received two doses of ILA (1 wk apart) are also shown (*).

 
We next considered the possibility that the memory cells in the lungs may circulate slowly, and that the splenic DCs cross-presenting ILA-loaded cells may not persist long enough to affect a significant proportion of this population. It has been estimated that the half-life of DCs is only 7–10 days in vivo (31). Therefore, to extend the time of potential exposure to tolerizing DCs, after 1 wk, mice were given a second dose of ILA-loaded apoptotic {beta}2M KO spleen cells. However, this extended treatment also did not decrease the numbers of HA memory cells in the lungs (Fig. 4B).

The effect of tolerance on the magnitude of a secondary response to viral infection

Upon secondary viral infection, reactivation and clonal expansion of memory T cells in lymphoid tissue results in the production of new effector cells that migrate to nonlymphoid tissues (32). Considering that the numbers of memory cells in parenchymal tissue are small compared with those found in the spleen (Figs. 1C and 4A), it would be anticipated that subsequent to secondary infection, the majority of effector cells in tissue would represent the progeny of lymphoid memory cells. Therefore, it would be anticipated that by successfully reducing the pool of lymphoid memory cells, tolerance would also reduce the numbers of effectors present in tissues following secondary challenge. To evaluate the influence of tolerance induction of memory CD8 T cells on the response to secondary viral infection, both the peptide-tolerized and control groups of mice were challenged with recombinant vaccinia virus expressing the NP protein, and the percentage and number of NP-specific T cells were assessed in various tissues. As shown in Fig. 5, A and B, there was a significant reduction in the numbers of IFN-{gamma}+ CD8 cells in all tissues of the mice that received tolerogenic peptide 4 wk before challenge. As summarized in Fig. 5C, the response was reduced by 70–80% in all tissues. This reduction also was evident in the CNS. This was presumably due to deletion by peptide of memory T cells in the spleen and lymph node, which would lead to a significantly reduced number of memory cells that are the precursors to the cells that migrate into the CNS during the secondary viral challenge. The efficiency of depletion was similar at early and later time points (days 4 and 10) following the secondary infection with vaccinia-NP (data not shown).



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  		FIGURE 5. Tolerance of memory T cells by soluble peptide or cross-presented Ag reduced the T cell response to a secondary viral infection. A–C, Forty days following PR8 infection, groups of mice (three mice/group) received either soluble NP peptide (100 µg, i.v.) (referred to as PR8 + NP peptide group) or HBSS (PR8 group). Thirty days later, both groups were infected with recombinant vaccinia virus expressing the NP gene (Vac-NP). Mice were sacrificed on day 6 following Vac-NP infection, and the percentage of NP-specific CD8+ T cells was assessed by intracellular staining for IFN-{gamma}. Results are representative of one of two experiments with similar results. The absolute numbers of NP-specific CD8 memory T cells (B), and percent depletion in each tissue (C), was calculated. D, Forty days following PR8 infection, one group of three mice received two doses of ILA-loaded {beta}2M KO spleen cells (30 x 106, i.v.) separated by a 1-wk interval, whereas the control group was untreated. Thirty days after the last injection of ILA-loaded cells, mice were infected with recombinant vaccinia virus expressing the KdHA epitope. Both groups of mice were sacrificed on day 6 after Vac-KdHA infection, and lymphocytes were isolated from different organs and the percentage of HA-specific CD8 T cells in each tissue was assessed by intracellular staining for IFN-{gamma}. Data represent the percent depletion of HA-specific CD8 cells from each organ obtained in one of two experiments, each with similar results.

 
It was also of interest to evaluate the influence of cross-tolerance of lymphoid-resident memory CD8 T cells by ILA-HA on the response to secondary viral infection. Following the induction of memory cell tolerance with ILA-loaded {beta}2M KO spleen cells, mice were challenged with recombinant vaccinia virus expressing the HA epitope, and the percentage and number of HA-specific T cells were assessed in various tissues. As shown in Fig. 5D, there was a significant reduction in the numbers of IFN-{gamma}+ CD8 cells in all tissues of the mice that received cross-presented tolerogen 4 wk before challenge. The response was diminished by ~80% in all tested tissues, including the spleen, lung, and liver. Therefore, although cross-presented tolerogen did not directly delete tissue-resident memory T cells, the tolerogen was indirectly able to reduce the numbers of tissue-resident effectors after secondary challenge by depleting the pool of lymphoid memory cells that serve as precursors to the effectors found in parenchymal tissue following secondary challenge.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have examined the tolerance susceptibility of CD8 memory T cells to both systemically delivered soluble peptide and Ag systemically cross-presented by quiescent DCs. In agreement with our previous results, we found that memory cells within secondary lymphoid tissue were readily eliminated by peptide. Furthermore, with the exception of cells within the CNS, most tissue-resident memory cells were also eliminated following exposure to soluble peptide.

T cells in the CNS may resist tolerance by soluble peptide for several different reasons. First, peptide would have great difficulty traversing the blood-brain barrier to enter the CNS. Second, even if peptide became available, such as when delivered by i.c.v. injection, under noninflammatory conditions, APCs in the CNS are sparse, and the lack of expression of MHC class I molecules on neurons (33) may further contribute to the failure of tolerance induction. Our finding that systemic soluble peptide was unable to tolerize memory cells in the CNS suggests not only that memory CD8 cells were refractory to tolerance within this privileged site, but also that they did not freely leave the CNS. Cells that left the CNS would have been tolerized by soluble peptide, thereby permanently reducing the numbers in the CNS. This observation is in agreement with results recently reported by Klonowski et al. (34), in which they used parabiosis to examine the migratory dynamics of blood-born memory cells between an animal harboring memory T cells and a nonimmune mouse. They found rapid equilibration of memory cells between most tissues in the donor and recipient, with the exception of the brain and intestinal mucosa. Therefore, it is likely that long-lived memory T cells in the CNS represent a relatively isolated subset that enter the tissue as effector cells soon after infection and become trapped, presumably due to the loss of expression of integrins that are required to migrate to other sites.

It is of interest to note that the majority, but not all memory cells were eliminated following exposure to a single bolus of soluble peptide. We have observed that most of the cells that remained were successfully deleted when the mice were given a second bolus of peptide 1 mo later. Therefore, the cells are not inherently refractory to deletion by peptide. These data are consistent with results in which we found that multiple exposures to peptide were required to achieve total deletion of naive TCR transgenic CD8 T cells (35). In those studies, which examined deletion vs anergy of a homogeneous population of naive TCR transgenic CD8 cells, it was determined that a certain proportion of the cells were anergized rather than deleted as the result of exposure to a high concentration of cognate peptide. These cells resisted further deletion until after Ag was cleared, and they could return to a responsive state. It is possible that, although the majority of memory cells were eliminated by a single exposure to peptide, the surviving cells may represent a population that was anergized rather than deleted. The difference between cells that are successfully deleted upon initial exposure to Ag and those that undergo anergy is not known.

Our results involving tolerance with cross-presented ILA-HA suggest that the majority of the tissue-resident memory cells do not gain access to cross-presented Ag. The simplest interpretation of these data is that the tissue-resident memory cells present at 1 mo following infection are unable to leave the tissue and therefore do not gain access to spleen or lymph nodes. This is consistent with the results of Hogan et al. (36), who showed that large numbers of tissue-resident cells remain static in the tissue, as well as recent reports which show that CCR7– T cells (the phenotype exhibited by the majority of tissue-resident memory T cells (37)) are unable to leave peripheral tissue (38, 39). However, our data are also consistent with an alternative possibility. It has been shown that the CD8{alpha}+ lymphoid DCs are the major population of APCs that are capable of cross-presenting Ags from captured dying cells (17, 40) and that this subset of DCs is mainly localized in the T cell-rich areas of the periarteriolar lymphatic sheath in the white pulp of the spleen (41, 42). It is possible that tissue-resident memory cells may enter the circulation but are confined to a migratory pathway that excludes their interaction with cross-presenting DCs in lymphoid tissue. The existence of such a migratory difference between different population of memory cells was suggested by the work of Weninger et al. (43), who found differential distribution of memory cells in the spleen, depending upon whether they were generated in vitro in the presence of IL-2 or IL-15. Whereas the cells generated with IL-15 exhibited characteristics of central memory cells inasmuch as they expressed high levels of CD62L and could freely enter lymph nodes and spleen, the cells generated with IL-2 exhibited many of the properties of tissue-resident memory cells. They were CD62Llow and could freely enter blood and spleen, but not lymph nodes. Of interest, although the IL-15-generated cells were confined to T cell areas in the spleen, the memory cells generated with IL-2 demonstrated a more diffuse pattern of localization and were also found within the red pulp and B cell zones. Also consistent with this observation, Unsoeld et al. (44) demonstrated that constitutive expression of CCR7 on effector CD8 T cells enabled these T cells to localize to the splenic white pulp, whereas CCR7– effector cells were excluded from the white pulp and accumulated in the area of the red pulp. Therefore, even if the CD62LlowCCR7– tissue-resident effector memory T cells could recirculate through the spleen, it is likely they would be unable to access the periarteriolar lymphatic sheath to find cross-presented Ag.

In addition, our data further suggest that the majority of tissue-resident memory T cells are a stable population that do not become central memory T cells. Any cells that so converted would have gained access to cross-presented Ag and been eliminated. This was found not to occur, even when mice were provided with two doses of ILA-HA. This result is in agreement with the recent finding of Marzo et al. (45) who found that, at low precursor frequency, effector memory T cells do not differentiate into central memory T cells.

The fact that deletion of memory T cells in the periphery can lower the number of T cells in the CNS following secondary viral infection has implications for treatment of autoimmune disease. For example, multiple sclerosis normally commences with a relapsing-remitting phase, later followed by a more progressive course (46, 47). Considering that the cervical lymphatics provide a communication bridge between the brain and the secondary lymphoid tissue (48), it is likely that decreasing the numbers of memory T cells residing in both the cervical lymph nodes and the spleen can dramatically reduce the pool of memory cells that can, upon secondary activation, proliferate and differentiate into effector T cells that infiltrate the CNS. Thus, although peripheral systemic delivery of soluble Ag may not delete memory autoreactive T cells within the CNS, it may still mitigate further CNS damage by reducing the influx of activated T cells from the periphery during a relapse. This mechanism of peripheral tolerance of pathogenic T cells by injection of soluble peptide may at least partly explain the prevention or amelioration of experimental autoimmune encephalomyelitis observed in animal model following systemic administration of myelin basic protein-derived peptides (49, 50, 51).

Similarly, despite the fact that cross-presented Ag could not tolerize tissue-resident memory cells, it was highly effective in reducing the numbers of effector cells in the tissue following secondary infection. Lymphoid memory T cells are the precursors of the majority of effector cells that are produced and disseminate in a relatively unrestricted manner following secondary challenge (32). Therefore, by reducing the numbers of memory cells in the spleen, cross-presented Ag was able to reduce the magnitude of the secondary response and the numbers of effectors in all tissues. This observation may have important implications for designing therapeutic strategies to mitigate T cell-mediated tissue destruction in autoimmune diseases or allograft rejection.

In summary, we have found that tissue-resident memory cells are highly susceptible to tolerance induction, but that both the form of tolerogen and location of the T cells can determine their accessibility to tolerogen and the degree to which they are deleted of from specific tissues. However, because both soluble and cross-presented forms of tolerogen are highly effective in eliminating memory cells in secondary lymphoid tissue, and because these serve as precursors to the large numbers of effectors that arise subsequent to secondary stimulation, either form of tolerogen can decrease considerably the numbers of effector cells that migrate to parenchymal tissue following secondary stimulation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 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 National Institutes of Health Grant DK50824-14. C.-H.W. was supported by a postdoctoral fellowship from the Whittier Institute for Diabetes. Back

2 Address correspondence and reprint requests to Dr. Linda A. Sherman, Department of Immunology, IMM-15, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: lsherman{at}scripps.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; HA, hemagglutinin; NP, nucleoprotein; {beta}2M, {beta}2-microglobulin; i.c.v., intracerebroventricular. Back

Received for publication June 15, 2005. Accepted for publication September 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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