Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0105007 on August 4, 2005

Published online before print August 4, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0105007v1
78/4/879    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kumaraguru, U.
Right arrow Articles by Rouse, B. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kumaraguru, U.
Right arrow Articles by Rouse, B. T.
(Journal of Leukocyte Biology. 2005;78:879-887.)
© 2005 by Society for Leukocyte Biology

In vivo rescue of defective memory CD8+ T cells by cognate helper T cells

Udayasankar Kumaraguru1, Kaustuv Banerjee and Barry T. Rouse1

Department of Microbiology, University of Tennessee, Knoxville

1Correspondence: M409 Department of Microbiology, University of Tennessee, Knoxville, TN 37996; Department of Microbiology, James Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614. E-mail: udayk@utk.edu and btr{at}utk.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The magnitude and efficacy of CD8+ T cell memory may notably regress, especially if immune induction occurs in the absence of adequate CD4+ help. This report demonstrates that this CD8+ memory malfunction could be remedied if a source of cognate antigen-recognizing helper cells were provided during recall. The inability of adoptive transfer of memory SIINFEKL-specific CD8 cells to reject tumors was overcome if recipients were primed for ovalbumin-specific helper cell responses. Additionally, animals primed for a SIINFEKL-specific memory response and incapable of rejecting the tumor could regain protective immunity if given helper cells. This pattern of CD8+ T cell functional rescue or reprogramming by helper cell transfer was replicated using a Herpes simplex virus antiviral immunity system. Our results could mean that therapeutic vaccine approaches could be designed to compensate situations that have defective CD8+ T cell function.

Key Words: vaccine • HSV


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The removal of numerous microbial agents and some tumors from the host depends principally on the function of CD8+ T lymphocytes [1 ]. Hence, one requirement for vaccine design against such diseases is that they induce optimal long-term CD8+ T cell immunity. Certain vaccine formulations may induce notable CD8+ responses in the acute phase, but the magnitude and functional efficacy decline dramatically with time [2 ]. For instance, immunization with peptides linked to adjuvants such as hsp70 or synthetic unmethylated CpG containing oligodeoxynucleotides may induce acute CD8+ responses comparable with live virus infection. However, the memory reactions were markedly inferior in magnitude and function to those accruing from virus infection [3 , 4 ]. Thus, memory CD8+ T cells induced by hsp70 peptide may show lower avidity than do memory cells resulting from live virus infection [3 ]. Reasons for the decline in magnitude and function of CD8+ T cell responses remain to be established.

One explanation, however, may relate to the need for adequate concomitant helper T cell (Th) activation during CD8+ T cell induction. In support of this, several groups have recently shown that if CD8+ T cells were induced in the absence of sufficient help, then long-term memory CD8+ T cell responses were inferior [5 6 7 8 9 ]. The helper effect could be replaced by stimulation with monoclonal antibodies to CD40, present on the CD8+ T cells themselves [10 ] or indirectly, by effects on antigen-presenting cells (APC) [11 ]. It is now widely accepted that adequate long-term memory CD8+ T cell immunity requires concomitant helper cell activation during immune induction, explaining why peptide immunization with CD8+ epitope peptides is usually unsatisfactory [12 ]. Less is understood about the role of Th cells at other phases of the CD8+ T cell immune response, such as differentiation of effectors into the long-term memory population or for the maintenance of long-term CD8+ memory. Moreover, it is not clear if defective memory CD8+ responses can be rescued to become more efficacious by re-exposure to Th cell stimulation. We have addressed this latter issue in the present communication. Our results demonstrate that the functional immune competence of memory CD8+ T cells can be improved by exposure to cognate helper cells in vivo. Rescue was achieved most effectively by CD4+ Th cell subsets, which produced interferon-{gamma} (IFN-{gamma}) rather than interleukin (IL)-2. The implication of these results for therapeutic vaccine design is discussed briefly.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Four- to five-week-old C57BL/6 (H-2b) mice were purchased from Harlan Sprague-Dawley (Indianapolis, Ind.). OT-I transgenics (Tg) were obtained from Dr. Michael Bevan (University of Washington, Seattle). OT-II Tg were provided by Dr. Brian Evavold (Emory University, Atlanta, GA). In conducting the research described in this work, we adhered to the Guide for the Care and Use of Laboratory Animals. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care.

Peptides
Herpes simplex virus (HSV)-gB [amino acids (aa) 498–505] peptide SSIEFARL, chicken ovalbumin (ova) class I (aa 257–264 SIINFEKL), and chicken ova class II (aa 323–339 ISQAVHAAHAEINEAGR) were synthesized and supplied by Research Genetics (Huntsville, AL).

Virus
HSV-1 KOS and HSV-1 17 were grown on Vero cell monolayers (American Type Culture Collection, Mansassas, VA, catalog No. CCL81), titrated, and stored in aliquots at –80°C until used.

Proteins
Recombinant hsp70 (rhsp70) was purchased from Stressgen Biotechnologies (Victoria, BC, Canada, catalog No. SPP-755). Dr. Jeffrey B. Ulmer (Chiron Corp., Emeryville, CA) provided HSV-gB protein.

Cell lines
The following cell lines were used: Vero (African green monkey kidney cell line), MC38 (C57BL/6, colon carcinoma, H-2b), and EMT6 (BALB/c mammary adenocarcinoma cells, H-2d). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY), supplemented with 10% heat-inactivated fetal bovine serum, 100 U penicillin G/ml, 100 µg streptomycin sulfate/ml, and 2 mM L-glutamine.

hsp70 and peptide binding
The peptides were loaded onto the hsp70 by the procedure described earlier [3 ]. In brief, the peptides were incubated with rhsp70 in binding buffer [phosphate-buffered saline (PBS) with 2 mM MgCl2] at 37°C for 60 min. Then, 0.5 mM adenosine 5'-diphosphate (Sigma Chemical Co., St. Louis, MO) was added, and the incubation was continued for another 60 min at the same temperature. As a control for carrier, peptides were complexed to bovine serum albumin by glutaraldehyde conjugation.

Immunizations
C57BL/6, OT-II, and OT-I mice were immunized with chaperone-peptide complex or with binding buffer only. The immunizations were done intraperitoneally (i.p.) on Days 0 and 21. Samples for acute-phase and memory-phase analysis were collected on Day 28 [7 days postinfection (p.i.)] or Day 81 (60 days p.i.), respectively. The i.p. route was chosen after experimenting with intramuscular and footpad injections. Ultraviolet-inactivated (uv)HSV-KOS (1x106) was injected i.p. in mice belonging to the virus control group.

Cytolytic T lymphocyte (CTL) assays
The CTL assay was performed as described earlier [13 ]. Briefly, effector cells generated after in vitro expansion (with peptide or HSV) were analyzed for their ability to kill major histocompatibility complex (MHC)-matched antigen-presenting targets. The targets included 51Cr-pulsed MHC-matched HSV-infected (MC38-HSV), MHC-matched SSIEFARL-pulsed (MC-38-SSIEFARL), MHC-mismatched HSV-infected and irrelevant peptide-pulsed (EMT6-HSV and EMT6-irrelevant peptide), and MHC-matched uninfected or unpulsed (MC-38) targets. The chromium release results were computed and are expressed as lytic units (LU)/106 as described elsewhere [3 ].

Intracellular IFN-{gamma} staining [intracellular cytokine staining (ICS)]
To enumerate the number of IFN-{gamma}-producing cells, ICS was performed as described previously [3 ]. In assays involving kinetics, peptide dose was varied from 5 µM to 0.01 µM.

Tetramer staining and flow cytometry
MHC class I (H-2d) tetramers to measure SSIEFARL- or SIINFEKL-specific T cells were provided by the NAIAD MHC Tetramer Core Facility (Atlanta, GA). A total of 106 cells obtained from these mice was stained with a mixture of fluorescein isothiocyanate (FITC)-labeled anti-CD8 (Caltag, South San Francisco, CA) and phycoerythrin (PE)-labeled tetramers for 45 min at 4°C. The controls included isotype control, stained cells, and unstained cells. They were then analyzed by using a FACScan machine and CellQuest software. The percentage values shown are the double-positive cells [CD8+ and peptide-specific T cell receptor (TCR)].

Enzyme-linked immunospot (ELISPOT) assay
The ELISPOT assay was used for quantification of cytokine-producing cells. ELISPOT plates (Millipore, Molseheim, France) were previously coated with IFN-{gamma} anti-mouse antibody. The immune T cells (responder cells) were mixed with syngeneic splenocytes (stimulator cells) pulsed with relevant or irrelevant peptide. Coincubation of the responder and stimulator cells was continued for 72 h at 37°C. The ELISPOT plates were washed three times with PBS and three times with PBS-Tween 20, and then biotinylated IFN-{gamma} antibody was added to the plates for 1 h at 37°C. The spots were developed using nitro blue tetrazolium (Sigma Chemical Co.) and 5-bromo-4-chloro-3-indolylphosphate (Sigma Chemical Co.) as a substrate following incubation with alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA) for 1 h and counted 24 h later under a stereomicroscope.

In vivo assay for CTL avidity measurement
Splenocytes from naive mice were stained with PKH26 (PKH26-GL, Sigma Chemical Co.) or 2.5 µM or 0.25 µM carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Eugene, OR). The CFSE-labeled cells alone were coated with relevant peptide (1 µM) or irrelevant peptide, and the PKH26-stained cells were used as peptide-unpulsed control. They were than transferred intravenously (5x106 cells of each population) into the indicated groups of mice. Five hours later, lymphocytes were isolated from spleen, as described previously. Target cells were distinguished from recipient cells based on PKH26 and CFSE staining. Dot plot was used to calculate the number of recovered PKH26-labeled peptide-unpulsed targets. Histogram plots were used to demonstrate the difference in separation pattern based on intensity of CFSE staining. The recovery and percent killing of the various CFSE-labeled, peptide-pulsed targets were calculated as follows: 100 – {[(% peptide pulsed in immunized/% unpulsed in immunized)/(% peptide pulsed in uninmmunized/% unpulsed in uninmmunized)]x100}.

Separation of IFN-{gamma}- or IL-2-producing CD4+ helper cells
To obtain purified population of IFN-{gamma} or IL-2 cytokine-secreting CD4+ helpers, we used the kit manufactured by Miltenyi Biotec (Auburn, CA). The procedure was as provided by the company. In brief, OT-II mice immunized previously with the optimum dose (2.5 µg) or four times the optimum dose (10 µg) were used as donors. Spleens were obtained from these mice, and single-cell preparations were restimulated for 16 h (IFN-{gamma}+ CD4+ Th isolation) or 6 h (IL-2+ CD4+ Th isolation) with specific peptide. Subsequently, an IL-2- or IFN-{gamma}-specific catch reagent was attached to the cell surface of all leukocytes. The cells were then incubated for a short time at 37°C to allow cytokine secretion. The secreted cytokine binds to catch reagent (IL-2 or IFN-{gamma}) on the positive cells. These cells can subsequently be labeled with appropriate second cytokine-specific antibody (mouse IL-2 or IFN-{gamma} detection antibody) conjugated to PE. The cytokine-secreting cells can now be magnetically labeled with anti-PE microbeads and enriched over a magnetic cell sorter (MACS) column, which was placed in the magnetic field of a MACS separator. The magnetically labeled cells were retained in the MACS column, and the unlabeled cells run through. The column was removed from the magnetic field, and retained cells were eluted as a positively selected cell fraction. The washed cells were then used for adoptive transfer studies. The frequency of IFN-{gamma}- and IL-2-producing CD4+ Th cells was ascertained by intracellular staining for the appropriate cytokine. Exposure to high-level antigen resulted in a skewing toward dominant IFN-{gamma}-secreting CD4+ Th cells, and optimum level resulted in predominately IL-2-producing cells. The profile of the memory CD4+ T cells, which were obtained, is as follows: high-dose antigen-stimulated IFN-{gamma}+: 76%; IL-2+: 6%; IFN-{gamma}/IL-2+: 9%; low-dose antigen-stimulated IL-2+: 60%; IFN-{gamma}+: 13%; IL-2/IFN-{gamma}+: 15%.

Adoptive transfers
Acute and memory T cells were generated in OT-I Tg or OT-II Tg or HSV-gB Tg or wild-type B6 mice, as described previously. Single-cell suspensions of pooled spleen cells were prepared from these mice. CD8+ T cells were shown to be ~89% SIINFEKL tetramer-specific and ~91% SSIEFARL tetramer-specific by flow cytometry in OT-I and gB Tg mice, respectively. Approximately 65% of OT-II CD4+ T cells were IFN-{gamma}-positive by intracellular staining after stimulation with ova class II peptide. ELISPOT assays were also done on donor populations before transfer. Various preparations in 0.2 ml PBS were injected into the tail vein of appropriate mice. Two days after transfer, the mice were used for subsequent analysis.

Tumor challenge experiments
Tumor challenge experiments were performed as described previously [14 ]. Briefly, mice received subcutaneous injections in the neck scuff with the ova gene-transfected tumor cell line EG7.Ova or its control parent EL4 tumor cell line, MC38, or MC38 pulsed with 2 mM SSIEFARL peptide. Ten–15 days after the tumor challenge. Mice were killed, and tumor growth was assessed by measuring the diameter of the tumor in millimeters (recorded as the average of two perpendicular diameter measurements). Mice that became moribund were killed.

Statistical analysis
The data were analyzed by dependent-sample t-test (comparison between two groups) or one-way ANOVA (Duncan; comparison of more than two groups) using SPSS for Windows, release 12.0 (SPSS, Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Rescue of CTL-mediated immunity by Th cells
As reported earlier, immunization with epitope peptides bound to hsp70 induces optimal acute CD8+ T cell responses, but the magnitude and avidity of memory reactivity were inferior to that induced by other forms of immunization [3 ]. To further analyze this issue, OT-I mice (TCR Tg mice with CD8+ T cells that recognize SIINFEKL) were primed with hsp70 coupled to SIINFEKL, a procedure that resulted in an average 89% of splenic CD8+ T cells being Tet+ CD62Llo in the acute (7 days post-prime) phase, and ~70% were CD8+ Tet+ CD44hi cells in the memory (60 days post-prime) phase. Splenocytes from OT-I hsp70 SIINFEKL-immunized (acute or memory) animals were adoptively transferred to B6 recipients, which themselves had been preimmunized 7 days previously in various ways. These included hsp70-ova 323–339 (to prime ova-specific CD4+ helper cells), keyhole limpet hemocyanin (KLH)-complete Freund’s adjuvant (CFA), or hsp70 alone or were nonimmunized. All groups were challenged 1 day after transfer with a tumor-producing EG7 thymoma cell (protection against this tumor is mediated by SIINFEKL-specific CD8+ T cells [14 ]). The results (Table 1 ) show that mice in all groups developed tumors, except for those in which Th cells had been primed and those that received acute ova-primed OT-I cells. Primed ova-specific helper cells alone failed to provide protection. These results indicate that the protective effect of ova-primed OT-I memory cells is nonfunctional but could be rescued by cognate ova-primed helper cells.


View this table:
[in this window]
[in a new window]
  		Table 1. Memory CTL Needs Helper T Lymphocyte Function to Achieve Tumor Protection

 
In additional experiments, OT-I Tg mice were primed with hsp70 SIINFEKL and subsequently adoptively transferred with OT-II T cells from naïve or from mice that had been primed with the cognate ova peptide (hsp70-ova 323–339). Two days after adoptive transfers, the mice in various groups (see Table 2 ) were challenged with EG7 tumor cells and were also tested for in vivo SIINFEKL-specific CTL activity. Again, the results show that acute-phase SIINFEKL-specific CD8+ T cells protected against tumor challenge, whereas memory cells did not. However, their functional activity could be rescued in the memory phase by reconstituting the animals with ova-specific CD4+ T cells. It is interesting that the acute-phase helper cells were effective immediately after transfer, as evidenced by enhanced killing in the in vivo CTL challenge assay. However, the memory-phase helper cells were better when given time to reactivate and provided functional restoration to CD8+ T cells, as demonstrated in the tumor challenge model. This probably explains the difference observed between in vivo CTL challenge and tumor challenge.


View this table:
[in this window]
[in a new window]
  		Table 2. Activation Status and Phenotype of Adoptively Transferred OT-II Cells Impact the CD8+ CTL Response

 
Cytokine-producing status of rescue Th cells
The above results indicated that the memory function of SIINFEKL-specific CD8+ cells can be rescued by CD4+ antigen-specific T cells. To determine if the helper cell effect was mediated preferentially by a subset of helper cells, OT-II mice were primed with hsp70-ova 323–339. At the acute (7 days post-prime) and memory stages (60 days post-prime), splenocytes were collected, stimulated in the presence of antigen for varying periods, as mentioned in Materials and Methods, and fractionated using MACS cell-enrichment columns into IL-2- and IFN-{gamma}-producing cell populations. Such cells were then adoptively transferred into memory-primed OT-I mice (as described previously). Two days post-transfer, animals were challenged with EG7 tumor cells or were tested for their SIINFEKL-specific in vivo CTL activity. The results (see Table 3 ) demonstrate that IL-2- and IFN-{gamma}-producing helper cell subsets could rescue SIINFEKL-specific anti-EG7 function in the acute phase, but only the IFN-{gamma}-producing CD4+ was effective in the memory phase. The possible reason for this observation may be that during acute phase, a good majority of the CD4+ Th cells may secrete both the cytokines. Furthermore, the in vivo CTL assay showed that the IFN-{gamma}-producing subset significantly heightened activity compared with recipients of the IL-2-producing Th cells (Fig. 1 ).


View this table:
[in this window]
[in a new window]
  		Table 3. Role of CD4+ Cytokine-Producing Subsets to Provide Help in Recalling CD8+ T Cell Responses

 


View larger version (28K):
[in this window]
[in a new window]
  		Figure 1. OT-II CD4+ IFN-{gamma}+ memory helper cells are better in rescuing the OT-I CD8+ CTL responses. OT-II Tg CD4+ T cells (5x106) were adoptively transferred on Day 0 to OT-I mice, which were immunized previously, and were in acute or memory phase. These transferees were taken from donor OT-II mice immunized previously with hsp70-ova class II peptide and in various stages of activation (effector or memory). They were unfractionated or separated into IFN-{gamma} or IL-2 producers using MACS separation columns after appropriate stimulation with cognate peptide.Exposure to high-level antigen resulted in a skewing toward dominant IFN-{gamma}-secreting CD4+ Th cells, and optimum level resulted in predominately IL-2-producing cells. Controls included naïve OT-I given PBS alone. Two days after transfer, the mice were challenged with peptide-pulsed targets (CFSElo) or peptide-unpulsed targets (CFSEhi) in an in vivo CTL assay. The spleens from in vivo CTL-challenged mice were harvested 5 h later and processed for flow cytometry analysis. The irrelevant peptide-pulsed targets recovered were taken to be as 100%. The experiment was repeated twice with similar results. Panels a–c: memoryhelper recipients as indicated; panels d, e: acute helper T cell recipients; f: no transfers received. The histogram represents data obtained from a single mouse in each group. However, the number in percentage indicates average lysis percentage from three mice. The difference among a, b, and c was [ANOVA (Duncan)] significant (b vs. c=P<0.0006; a vs. b=P<0.0705; a vs. c=P<0.0187).

 
Rescue of antiviral CD8+ function by Th cells
The relevance of the ova-specific CD8+ memory rescue experiments was extended to a viral system in which the predominant CD8+ T cell-protective immune response is accounted for by activity against the gB-derived peptide SSIEFARL [15 ]. In this system, previous results had shown that immunization with hsp70 peptide in the absence of concomitant help generated defective peptide-specific memory responses [3 , 4 , 16 ]. To determine if such defective memory could be rescued, groups of B6 mice were primed with hsp70 SSIEFARL and then in the memory phase (Day 60 post-priming), were given adoptive transfers of column-purified CD4+ T cells. The CD4+ T cell donors were immunized with gB protein + CFA and taken at Day 7 or 45 post-immunization. Naïve B6 CD4+ T cells, as well as ova 323–339 peptide-primed OT-II CD4+ T cells were used as additional controls. Two days after transfer, the various groups were tested for in vivo CTL activity using targets expressing the SSIEFARL peptide or tested for CD8+ T cell functional immunity. Two assays were used to measure the latter. These were protection against viral challenge using the zosteriform model [17 ] and tumor challenge by SSIEFARL-expressing MC38 tumor cells. The pattern of results observed closely parallels those observed in the ova-specific system. Thus, whereas acute gB-specific CD8 provided protection against viral and tumor challenge and expressed in vivo CTL activity, memory cells were without significant activity. Nevertheless, functional activity could be rescued by antigen-specific but not by nonspecific Th cell transfers, as shown in Table 4 . Antigen-specific helpers could provide the helper cell rescue effect, whether these were antigen-primed in the acute or memory phase. As measured by an in vivo CTL assay (Fig. 2 ), however not significant, the memory-phase helper cell-adoptive transfers rescued notably higher CTL activity than was evident with acute-helper cell-adoptive transfers. This could be explained by the fact that the antigen-specific memory helper cells, activated briefly in vitro, are much more efficient in rescuing CD8+ T cell function. Results from further experiments (varying the number of adoptively transferred cells) also indicated that the frequency of gB-specific CD4+ helper was not enough to afford protection by themselves. The same could be said for explaining the limited role the naïve cell transfer plays in affording protection, as ova and HSV might stimulate the reconstituted, naïve CD4+ Th cells. Additionally, the immunization protocol involved in generating the cognate CD4+ Th cells used in transfers is no way comparable with stimulation they get during challenge.


View this table:
[in this window]
[in a new window]
  		Table 4. CD8 CTL Recall Response to HSV Is Enhanced by the Presence of Memory Th Cells

 


View larger version (31K):
[in this window]
[in a new window]
  		Figure 2. Help during recall reprograms antiviral CD8+ CTL responses. On Day 0, 5 x 106 CD4+ (column-purified) donor T cells, obtained from gB protein + CFA immunized mice and in various stages of activation (naïve, effector, or memory), were adoptively transferred to C57BL/6 mice immunized previously with hsp70 SSIEFARL. In addition, OT-II effectors were transferred to a group as noncognate helper controls. Two days after transfer, the mice were challenged with peptide-pulsed or unpulsed targets, differentially labeled with CFSE (high and low, respectively) in an in vivo CTL assay. The controls for these included irrelevant peptide (SIINFEKL)-pulsed targets and peptide-unpulsed PKH-26-labeled cells. The experiment was repeated three times with a similar pattern of results. (a) Naive CD4 recipient; (b) memory CD4 recipient; (c) acute CD4 recipient; (d) no transfers received; (e) naive control; (f) OT-II CD4 (noncognate) transfer. The histogram represents data obtained from a single mouse in each group. However, the number in percentage indicates average lysis percentage from three mice. The difference between b and c was not statistically significant (P<0.0344).

 
In vitro characterization of rescued CD8+ T cells
The gB-specific CD8+ T cells, which were rescued in vivo, were isolated from mice 5 days after CD4+ T cell transfers and subjected to further in vitro analyses. First, as a measure of change in avidity, the CD8+ T cells, obtained from mice that were reconstituted with helper cells or not, were tested for their ability to lyse peptide-pulsed target versus virus-infected targets in an in vitro CTL assay (Table 5 ). The differences in LU between the two targets (peptide-pulsed vs. virus-infected) were lowest in groups that were reconstituted with cognate helper cells (62 vs. 46, respectively) or were in acute phase of response (71 vs. 63, respectively). The ability to lyse peptide-pulsed as well as virus-infected targets efficiently indicated their higher avidity status. This was, however, not observed in groups that were given naïve CD4+ T cells or OT-II CD4+ T cells. Second, the CD8+T cells from these mice were tested in vitro for functional rescue by stimulating them with varying concentrations of cognate peptide (SSIEFARL) and subsequently stained for intracellular IFN-{gamma}. This assay measures not only cytokine production but also the frequency of the responding population. As is evident in Figure 3 , the basal level of CD8+ T cells responding to the highest concentration of peptide (5 µM) in mice reconstituted with naïve CD4+ Th cells was only 2.8%. In contrast, the mice reconstituted with cognate memory CD4+ Th cells were ~13% at the highest peptide concentration; this was equivalent to the patterns observed in the mice that were in the acute phase of the response. However, the most interesting observation was that the reconstitution by noncognate OT-II memory CD4+ Th cells barely helped rescue of defective memory. The results not only demonstrate the increased magnitude of helper-assisted recall response but also their sensitivity to low concentrations of peptide to elicit a response compared with unhelped CD8+ T cells.


View this table:
[in this window]
[in a new window]
  		Table 5. CD8+ T Cells in gB-CD4+ Th Cell-Reconstituted Mice Show Enhanced Lytic Activity

 


View larger version (63K):
[in this window]
[in a new window]
  		Figure 3. Peptide dose-dependent increase in IFN-{gamma} response by CD8+ T cells is evident in rescued mice. The mice were reconstituted with cognate or noncognate CD4+ Th cells. Two days later, uvHSV1 (KOS; 1x104 pfu before uv inactivation) was given i.p. to recall anti-HSV responses. On Day 5, the mice were killed and analyzed for the ability of CD8+ T cells to make IFN-{gamma} in response to stimulation with the cognate peptide (SSIEFARL). The peptide dose was varied from 5 µM to 0.01 µM. The cells were stimulated for 6 h as mentioned in Materials and Methods and processed for surface and intracellular staining. The experiment was done on three different mice in each group at a given time-point. The figure represents the average obtained from three mice. The statistical significance was worked out with SPSS software. The difference between the group that received the cognate memory CD4+ Th cells and the noncognate memory CD4+ Th cells was statistically significant (P<0.001).

 
The differences in the patterns observed were statistically significant. In addition, CD8+ T cells obtained from rescued mice stained intensely with tetramers (data not shown), indicating their superior quality, as previously suggested by Oh et al. [18 ]. Taken together, these experiments with antiviral immunity confirm the pattern of events established with the ova-specific systems and demonstrate that functional CTL can be rescued by the provision of appropriate help after the immune-induction phase.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This report addresses the role of helper cells on influencing the in vivo function of memory CD8+ T cell-mediated immunity. Animals immunized with hsp70 peptide develop effective, acute CD8+ responses, but the memory reactions were limited and functionally inefficient [3 ]. Our results demonstrate that this memory malfunction could be remedied if a source of cognate antigen-recognizing helper cells were provided during recall. The inability of SIINFEKL-specific memory CD8+ T cells to reject EG7 tumor cells (that express SIINFEKL) could be restored if a source of helper cells reactive with ova were provided. This was shown in two ways. In the first approach, the inability of adoptively transferred memory SIINFEKL-specific CD8 cells to reject tumors was overcome if recipients were primed for ova-specific helper cell responses. Second, animals primed for a SIINFEKL-specific memory response and incapable of rejecting the tumor could regain protective immunity if given helper cells. This pattern of CD8+ T cell functional rescue by helper cell transfer was replicated using an antiviral immunity system. Our results mean that therapeutic vaccine approaches could be designed to compensate situations where CD8+ T cell function is defective.

With acute virus infections, as best exemplified by lymphocytic choriomengitis virus, CD8+ T cell memory remains robust indefinitely [19 ]. Alas, such is not the case for all infections. For instance, in human immunodeficiency virus (HIV), a dysfunction of CD8+ memory may occur, accounting in part for the decline in protective immunity [20 ]. In other persistent infections that include HSV, disease has also been associated with a decline in CD8+ T cell function [21 , 22 ]. Hence, there exists an interest in finding means of rescuing functional immunity, perhaps topping up resistance levels with appropriate therapeutic vaccines. Studies in vitro with HIV-specific CD8 T cells indicate that defective function can be overcome by forms of stimulation that induce IL-15 [20 ]. In our report, we demonstrate a novel role for helper cells to rescue CD8+ T cell function. The helpers could be recently primed or memory cells. However, the effect was only evident with helpers that could recognize specific antigen, although the source of the ova antigen was not obvious. Thus, in the case of the tumor protection experiments, the EG7 cells, although transfected with the ova protein gene themselves, only express the SIINFEKL peptide at their cell surface [23 ]. Hence, living cells would be invisible to ova 323–339 recognizing specific helper cells. We presume, however, that the source of stimulation could derive from dying EG7 cells reprocessed by host APC, as has been suggested by others [24 ], or the brief in vitro stimulation with the cognate period before transfer was suffice. Accordingly, mice immunized with EG7 cells developed immune responses characteristic of class II MHC-restricted T cells, including antibody responses [25 ].

Our results emphasizing the importance of cognate help have been supported by a recent observation by Smith et al. [26 ]. They demonstrated that help was essential for the generation of CTL immunity to HSV-1 and that the CD4+ T cells mediated help in a cognate, antigen-specific way. This study provided direct in vivo evidence for DC licensing by helper T cells and showed that licensing was rapid and essential for the formation of effector and memory CTL.

We have explored in this report the consequence of the phenotype of such helper cells and the mechanism by which the rescue effect was accomplished. By means of adoptive transfer experiments, we could show that different Th cell subsets acted differentially in rescuing the function of the antitumor CD8+ T cell response. Thus, when antigen-stimulated memory CD4+ helper cells were fractionated on affinity columns into IFN-{gamma} and IL-2 producers, the former population was more effective at providing rescue. This observation was unexpected, as one idea to explain the functional improvement of CD8 T cells was that IL-2 produced by the helper cells could be causing the CD8+ memory T cells to expand. Such an idea was advocated by the Schoenberger group [8 ] to explain the requirement for help in CD8+ T cell priming. In that situation, the absence of help could be largely overcome by the provision of exogenous IL-2 [8 ]. It remains to be shown how the IFN-{gamma}-producing helper cells affect rescue and whether IFN-{gamma} or some other molecule is involved in the process. Our speculation is that the CD8+ T cells modulate their signal transducers and activators of transcription and become more responsive to IFN-{gamma} than IL-2 in the memory phase. However, it has been demonstrated earlier that CD4+ T cells provide help to CD8+ T cells by secreting key cytokines, resulting in up-regulation of costimulatory molecules on APCs and B cells; deliver stimulatory signals to their CD40 ligand by CD40 expressed on APCs and B cells; or CD4+ T cells directly communicating with CD8+ T cells by T–T interaction mediated by CD40 again. In addition, the self-renewing capacity of the CD8+ T cell was reduced, and the numbers have been shown to decline in the absence of CD4+ T cells [9 , 19 , 27 , 28 ].

It was unknown whether secondary (recall) responses needed CD4 help until the work of Shedlock and Shen [7 ]. They showed that depletion of CD4 cells during the recall response had minimal effect on CD8 memory. However, in a recent report, Sun et al. [29 ] have provided evidence that the presence of CD4+ T cells during memory maintenance is more crucial to the health and survival of memory CD8+ T cells rather than during priming. Thus, it remains to be determined if the CD8+ T cells primed in the absence of help could be reprogrammed to behave differently to homeostatic signals or even to recall responses. The results from our study seem to indicate that such reprogramming occurred and rescued CD8+ T cells to an extent.

Although our study demonstrates that in vivo SIINFEKL-specific CTL function recalled from memory is facilitated by the presence of helper cells, we do not understand how the elevated response of the CD8+ T cells occurs. A number of possibilities are under investigation. These include increased proliferative capacity, changed balance between functional subsets of CD8+ T cells, as well as changes in overall avidity of CTL when recalled in the presence of helper cell coactivation (or reprogramming). Some information is available regarding the latter issue. Accordingly, the absence of significant difference in the increase in quantity of tetramer-specific CD8+ T cells (data not shown) among the groups after rescue seems to indicate changes to be qualitative. It is possible that reprogramming occurred during homeostatic turnover, resulting in improvement of their avidity. Thus, when the in vitro function of CTL from helper-deprived and helper-rescued cells was compared in vitro for their ability to kill virus-infected and peptide-sensitized target cells, differences were evident. Accordingly, the helper-assisted CTL population was judged to manifest higher avidity, as this population killed virus-infected and peptide-sensitized targets almost equally. In contrast, the nonrescued CTL population killed peptide targets more effectively than those sensitized with virus. Such a phenomenon was shown by others to be a correlate of low-avidity CTL [30 ]. The avidity issue is under further investigation.

Most of our studies were performed using the well-characterized ova system. However, most importantly, we were also able to show that defective memory CD8+-mediated immunity to HSV, resulting from hsp70-peptide immunization, could be overcome by transfer of viral antigen-specific helper cells to animals with defective CD8+ immunity. The consequence of such rescue was a marked increase in the potency of antiviral immunity. Such studies have particular significance for the future design of therapeutic vaccines. Accordingly, HSV is one of those many human pathogens for which a therapeutic vaccine is warranted. Although not proven, there is a strong suspicion that persons who suffer frequent clinical problems with herpes have subtle defects in T cell immunity, principally CD8+T cell responses [31 ]. Could it be that vaccines that expand appropriate helper cell subsets may provide the functional boost to CD8+ T cell-mediated responses accountable for curtailing infection? This issue warrants investigation.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grants AI14981 and AI46462. Additional support also came from Center for Excellence, USDA, College of Veterinary Medicine, University of Tennessee (Knoxville). Tetramers were provided by National Institute of Allergy and Infectious Diseases, MHC Tetramer Core Facility, Yerkes Regional Primate Research Center (Atlanta, GA). OT-I Tg mice were obtained from Dr. Michael Bevan, University of Washington (Seattle). OT-II Tg were provided by Dr. Brian Evavold, Emory University (Atlanta, GA). HSV-gB protein was a kind gift from Dr. J. Ulmer, Chiron Corporation.

Received January 7, 2005; revised June 19, 2005; accepted June 21, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wong, P., Pamer, E. G. (2003) CD8 T cell responses to infectious pathogens Annu. Rev. Immunol. 21,29-70[CrossRef][Medline]
  2. Zinkernagel, R. M. (2003) On natural and artificial vaccinations Annu. Rev. Immunol. 21,515-546[CrossRef][Medline]
  3. Kumaraguru, U., Gierynska, M., Norman, S., Bruce, B. D., Rouse, B. T. (2002) Immunization with chaperone-peptide complex induces low-avidity cytotoxic T lymphocytes providing transient protection against herpes simplex virus infection J. Virol. 76,136-141[Abstract/Free Full Text]
  4. Gierynska, M., Kumaraguru, U., Eo, S. K., Lee, S., Krieg, A., Rouse, B. T. (2002) Induction of CD8 T-cell-specific systemic and mucosal immunity against herpes simplex virus with CpG-peptide complexes J. Virol. 76,6568-6576[Abstract/Free Full Text]
  5. Bourgeois, C., Veiga-Fernandes, H., Joret, A. M., Rocha, B., Tanchot, C. (2002) CD8 lethargy in the absence of CD4 help Eur. J. Immunol. 32,2199-2207[CrossRef][Medline]
  6. Sun, J. C., Bevan, M. J. (2003) Defective CD8 T cell memory following acute infection without CD4 T cell help Science 300,339-342[Abstract/Free Full Text]
  7. Shedlock, D. J., Shen, H. (2003) Requirement for CD4 T cell help in generating functional CD8 T cell memory Science 300,337-339[Abstract/Free Full Text]
  8. Janssen, E. M., Lemmens, E. E., Wolfe, T., Christen, U., von Herrath, M. G., Schoenberger, S. P. (2003) CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes Nature 421,852-856[CrossRef][Medline]
  9. Kaech, S. M., Ahmed, R. (2003) Immunology. CD8 T cells remember with a little help Science 300,263-265[Abstract/Free Full Text]
  10. Schoenberger, S. P., Toes, R. E., van der Voort, E. I., Offringa, R., Melief, C. J. (1998) T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions Nature 393,480-483[CrossRef][Medline]
  11. Rocha, B., Tanchot, C. (2004) Towards a cellular definition of CD8+ T-cell memory: the role of CD4+ T-cell help in CD8+ T-cell responses Curr. Opin. Immunol. 16,259-263[CrossRef][Medline]
  12. Ahlers, J. D., Belyakov, I. M., Terabe, M., Koka, R., Donaldson, D. D., Thomas, E. K., Berzofsky, J. A. (2002) A push-pull approach to maximize vaccine efficacy: abrogating suppression with an IL-13 inhibitor while augmenting help with granulocyte/macrophage colony-stimulating factor and CD40L Proc. Natl. Acad. Sci. USA 99,13020-13025[Abstract/Free Full Text]
  13. Kumaraguru, U., Davis, I. A., Deshpande, S., Tevethia, S. S., Rouse, B. T. (2001) Lymphotoxin {alpha}–/– mice develop functionally impaired CD8+ T cell responses and fail to contain virus infection of the central nervous system J. Immunol. 166,1066-1074[Abstract/Free Full Text]
  14. Gao, F. G., Khammanivong, V., Liu, W. J., Leggatt, G. R., Frazer, I. H., Fernando, G. J. (2002) Antigen-specific CD4+ T-cell help is required to activate a memory CD8+ T cell to a fully functional tumor killer cell Cancer Res. 62,6438-6441[Abstract/Free Full Text]
  15. Wallace, M. E., Keating, R., Heath, W. R., Carbone, F. R. (1999) The cytotoxic T-cell response to herpes simplex virus type 1 infection of C57BL/6 mice is almost entirely directed against a single immunodominant determinant J. Virol. 73,7619-7626[Abstract/Free Full Text]
  16. Kumaraguru, U., Suvas, S., Biswas, P. S., Azkur, A. K., Rouse, B. T. (2004) Concomitant helper response rescues otherwise low avidity CD8+ memory CTLs to become efficient effectors in vivo J. Immunol. 172,3719-3724[Abstract/Free Full Text]
  17. Manickan, E., Rouse, B. (1995) Roles of different T-cell subsets in control of herpes simplex virus infection determined by using T-cell-deficient mouse-models J. Virol. 69,8178-8179[Abstract]
  18. Oh, S., Perera, L. P., Burke, D. S., Waldmann, T. A., Berzofsky, J. A. (2004) IL-15/IL-15R{alpha}-mediated avidity maturation of memory CD8+ T cells Proc. Natl. Acad. Sci. USA 101,15154-15159[Abstract/Free Full Text]
  19. Wherry, E. J., Ahmed, R. (2004) Memory CD8 T-cell differentiation during viral infection J. Virol. 78,5535-5545[Free Full Text]
  20. Ostrowski, M. A., Justement, S. J., Ehler, L., Mizell, S. B., Lui, S., Mican, J., Walker, B. D., Thomas, E. K., Seder, R., Fauci, A. S. (2000) The role of CD4+ T cell help and CD40 ligand in the in vitro expansion of HIV-1-specific memory cytotoxic CD8+ T cell responses J. Immunol. 165,6133-6141[Abstract/Free Full Text]
  21. Grakoui, A., Shoukry, N. H., Woollard, D. J., Han, J. H., Hanson, H. L., Ghrayeb, J., Murthy, K. K., Rice, C. M., Walker, C. M. (2003) HCV persistence and immune evasion in the absence of memory T cell help Science 302,659-662[Abstract/Free Full Text]
  22. Posavad, C. M., Koelle, D. M., Shaughnessy, M. F., Corey, L. (1997) Severe genital herpes infections in HIV-infected individuals with impaired herpes simplex virus-specific CD8+ cytotoxic T lymphocyte responses Proc. Natl. Acad. Sci. USA 94,10289-10294[Abstract/Free Full Text]
  23. Naylor, S., Ji, Q., Johnson, K. L., Tomlinson, A. J., Kieper, W. C., Jameson, S. C. (1998) Enhanced sensitivity for sequence determination of major histocompatibility complex class I peptides by membrane preconcentration-capillary electrophoresis-microspray-tandem mass spectrometry Electrophoresis 19,2207-2212[CrossRef][Medline]
  24. Inaba, K., Turley, S., Yamaide, F., Iyoda, T., Mahnke, K., Inaba, M., Pack, M., Subklewe, M., Sauter, B., Sheff, D., Albert, M., Bhardwaj, N., Mellman, I., Steinman, R. M. (1998) Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells J. Exp. Med. 188,2163-2173[Abstract/Free Full Text]
  25. Mowat, A. M., Donachie, A. M. (1991) Studies on the immunogenicity of an endogenously processed protein antigen in mice Immunol. Lett. 27,243-249[CrossRef][Medline]
  26. Smith, C. M., Wilson, N. S., Waithman, J., Villadangos, J. A., Carbone, F. R., Heath, W. R., Belz, G. T. (2004) Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity Nat. Immunol. 5,1143-1148[CrossRef][Medline]
  27. von Herrath, M., Yokoyama, M., Dockter, J., Oldstone, M., Whitton, J. (1996) CD4-deficient mice have reduced levels of memory cytotoxic T lymphocytes after immunization and show diminished resistance to subsequent virus challenge J. Virol. 70,1072-1079[Abstract]
  28. Belz, G. T., Wodarz, D., Diaz, G., Nowak, M. A., Doherty, P. C. (2002) Compromised influenza virus-specific CD8+-T-cell memory in CD4+-T-cell-deficient mice J. Virol. 76,12388-12393[Abstract/Free Full Text]
  29. Sun, J. C., Williams, M. A., Bevan, M. J. (2004) CD4+ T cells are required for the maintenance, not programming, of memory CD8+ T cells after acute infection Nat. Immunol. 5,927-933[CrossRef][Medline]
  30. Alexander-Miller, M. A., Leggatt, G. R., Berzofsky, J. A. (1996) Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy Proc. Natl. Acad. Sci. USA 93,4102-4107[Abstract/Free Full Text]
  31. Koelle, D. M., Posavad, C. M., Barnum, G. R., Johnson, M. L., Frank, J. M., Corey, L. (1998) Clearance of HSV-2 from recurrent genital lesions correlates with infiltration of HSV-specific cytotoxic T lymphocytes J. Clin. Invest. 101,1500-1508[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. L. Hwang, J. R. Lukens, and T. N. J. Bullock
Cognate Memory CD4+ T Cells Generated with Dendritic Cell Priming Influence the Expansion, Trafficking, and Differentiation of Secondary CD8+ T Cells and Enhance Tumor Control
J. Immunol., November 1, 2007; 179(9): 5829 - 5838.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0105007v1
78/4/879    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kumaraguru, U.
Right arrow Articles by Rouse, B. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kumaraguru, U.
Right arrow Articles by Rouse, B. T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS