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The Journal of Immunology, 2007, 178, 6350 -6358
Copyright © 2007 by The American Association of Immunologists, Inc.
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Requirement for CD4 T Cell Help in Maintenance of Memory CD8 T Cell Responses Is Epitope Dependent1

Elizabeth A. Ramsburg*, Jean M. Publicover*,{dagger}, Dagan Coppock* and John K. Rose2,*

* Department of Pathology and {dagger} Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06510


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD4 Th cells play critical roles in stimulating Ab production and in generating primary or maintaining memory CTL. The requirement for CD4 help in generating and maintaining CTL responses has been reported to vary depending on the vector or method used for immunization. In this study, we examined the requirement for CD4 T cell help in generating and maintaining CTL responses to an experimental AIDS vaccine vector based on live recombinant vesicular stomatitis virus (VSV) expressing HIV Env protein. We found that primary CD8 T cell responses and short-term memory to HIV Env and VSV nucleocapsid (VSV N) proteins were largely intact in CD4 T cell-deficient mice. These responses were efficiently recalled at 30 days postinfection by boosting with vaccinia recombinants expressing HIV Env or VSV N. However, by 60 days postinfection, the memory/recall response to VSV N was lost in CD4-deficient mice, while the recall response HIV Env was partially maintained in the same animals for at least 90 days. This result indicates that there are epitope-specific requirements for CD4 help in the maintenance of memory CD8 T cell responses. Our results also suggest that choice of epitopes might be critical in an AIDS vaccine designed to protect against disease in the context of reduced or declining CD4 T cell help.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recombinant vesicular stomatitis viruses (VSVs)3 expressing appropriate foreign Ags have been used to generate effective experimental vaccines protecting against infection or disease caused by multiple viral pathogens including an SIV-HIV hybrid causing AIDS in rhesus macaques (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). The VSV-based HIV vaccine developed in our laboratory has protected macaques from AIDS for up to 5 years postchallenge, but it does not generate sterilizing immunity (10). Vaccinated animals are infected with the challenge virus but then control their viral loads, often to levels below detection. However, some vaccinated animals had significantly reduced CD4 T cell counts following the SIV-HIV hybrid challenge (6, 10). Any effective HIV vaccine will likely be widely distributed and might also be used in a therapeutic context, for example, to stimulate anti-HIV immunity in individuals with reduced CD4 T cell counts undergoing combination drug therapy. This situation raises the issue of whether individuals in whom CD4 T cell counts are compromised either through the effects of HIV infection or other means could be effectively immunized.

Several detailed studies have demonstrated that while the primary expansion of antiviral CD8 T cells can occur independent of CD4 T cell help, CD4 help is required for the long-term (>2 mo) survival of memory CD8 T cells (12, 13, 14). The loss of nonhelped CD8 T cells is particularly apparent during chronic viral infection (15, 16, 17, 18). One mechanism of CD8 T cell loss was recently identified by Barber et al. (19), who demonstrated that overexpression of the proapoptotic receptor PD-1 on CD8 T cells primed in the absence of CD4 help leads to their elimination during chronic lymphocytic choriomeningitis virus infection.

Although it is clear that retention of memory CTL responses to several infectious agents is dependent on CD4 T cell help (20, 21, 22) (13, 14), it is not known whether the amount of help required differs according to Ag specificity. The experiments in this study were designed to examine the requirement for CD4 help in primary and memory CD8 T cell responses to vector and vaccine Ags in animals infected with a single VSV vector encoding the HIV Env protein.

The requirement for CD4 T cell help in primary and secondary antiviral CD8 T cell responses has been an area of considerable controversy. In the case of responses to VSV, it was reported initially that generation of primary cytotoxic CD8 T cell responses was strongly dependent on CD4 T cell help (23). Other studies reported little or no requirement for CD4 T cell help in such responses (24, 25). However, it is difficult to compare the results among these studies because different methodologies were used to eliminate CD4 cells and to analyze immune responses. The CD4 dependence of recall responses to VSV has not been examined directly.

In this study, we report that primary CD8 T cell responses to a VSV nucleocapsid (VSV N) epitope, as well as to an HIV Env epitope, are largely intact in CD4 T cell-deficient mice immunized with a rVSV encoding HIV Env. When we examined resting memory and recall responses to the VSV N epitope and the HIV Env p18 epitope in these mice, we found that the ability of the cells to re-expand differed dramatically between the two Ags. VSV N-specific CD8 T cells lost the capacity for re-expansion after 60 days postimmunization while Env-specific CD8 T cells would still expand after 90 days. Although nonspecific factors are undoubtedly involved in the maintenance of memory CD8 T cells, our results indicate there must also be Ag-specific factors involved as well. Our results also indicate that there could be a rational basis for choosing less CD4-dependent epitopes in design of an effective AIDS vaccine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Inoculation of mice

Mice were either obtained from The Jackson Laboratory or bred and maintained in our laboratory. To obtain CD4–/– mice expressing the MHC class I allele H-2d (BALB/c allele), C57BL/6 CD4–/– mice were crossed onto the BALB/c background. Mice were housed in microisolator cages in a biosafety level 2-equipped animal facility. Viral stocks were diluted to appropriate titers in serum-free DMEM. For i.m. vaccination, mice were injected with 5 x 105 PFU of virus in 50 µl. For i.p. vaccination, mice were injected with 1 x 105 PFU of virus in a 100-µl total volume. The Institutional Animal Care and Use Committee of Yale University approved all animal experiments.

Tetramer assay

Splenocytes were obtained by disrupting spleens between the frosted ends of two microscope slides. RBC were removed using RBC-lysing buffer (Sigma-Aldrich). To obtain lymphocytes from lungs, mice were perfused with sterile PBS until lungs were cleared of blood. The lungs were removed, chopped into fine pieces, and digested for 2 h at 37°C in DMEM containing 5% FCS, 150 U/ml collagenase, and 20 µg/ml DNase. After digestion, cells were mashed through a metal sieve, filtered, and layered onto a Ficoll gradient. Gradients were centrifuged (2000 rpm for 30 min at room temperature (RT)) in a Sorvall Legend RT centrifuge and lymphocytes were collected from the interface. Cells were washed and resuspended in DMEM containing 5% FCS. Staining was performed on freshly isolated lymphocytes. Briefly, ~5 x 106 cells were added to the wells of a 96-well V-bottom plate and were blocked with unconjugated streptavidin (Molecular Probes) and Fc block (BD Pharmingen) for 15 min at RT. Following a 5-min centrifugation at 500 x g, splenocytes were labeled with a FITC-conjugated anti-CD62L Ab (BD Pharmingen), an allophycocyanin-conjugated anti-CD8 Ab (BD Pharmingen), and tetramer for 30 min at RT. The N1 tetramer was a PE-conjugated MHC class I Dd tetramer (provided by Dr. L. Lefrancois, Farmington, CT) containing the VSV N1 peptide (N-RGYVYQGL-C;). The VSV N2 pentamer was provided by D. Cooper (Wyeth Pharmaceuticals, Pearl River, NY) and the HIV EnvP18 tetramer was provided by the National Institutes of Health AIDS Reagent facility. CD8 T cells which were tetramer positive and activated CD62Llow were identified by flow cytometry. Animals vaccinated with rVSV were used to determine background levels of tetramer binding. Background was routinely <0.1% and was subtracted from all reported percentages. No difference in binding or staining was observed in head-to-head comparisons of the tetramer and pentamer reagents (when a N2 tetramer and pentamer were compared).

CTL assay in vivo

This assay was performed as described previously (26) using Env peptide p18-I10 (N-RGPGRAFVTI-C; Invitrogen Life Technologies) or VSV N1 peptide (N-RGYVYQGL-C; Invitrogen Life Technologies). On day 7 postinoculation, splenocytes were obtained as described above from an uninfected mouse and resuspended in 1 ml of 5% FBS-DMEM. The donor (target) cells were split into two populations. Env p18-I10 or VSV N1 peptide was added to one population (+peptide) to a final concentration of 10–6 M, and to the other population no peptide was added (–peptide). Cells were incubated at 37°C in 5% CO2 for 45 min with occasional mixing. Cells were washed and resuspended in 1 ml of PBS. One milliliter of 10 µM CFSE (Molecular Probes) was added to peptide cells (final concentration, 5 µM) to generate a CFSEhigh group and 1 ml of 1 µM CFSE was added to peptide cells (final concentration, 0.5 M) to generate the CFSElow group. Cells were vortexed as the CFSE was added and then incubated for 5 min at RT. Cells were then washed three times in PBS and resuspended in PBS at a concentration of 108 cells/ml. Ten million cells (100 µl) were injected i.v. into vaccinated or control (immunized with rVSV) mice. After 4 h, the recipient mice were euthanized and spleens were obtained and prepared as above. CFSEhigh and CFSElow populations were identified by flow cytometry. Percent-specific lysis was calculated by using the following formula: percent-specific lysis = (1/(ratio for vaccinated mice/ratio for control mice)) x 100, where "ratio" = (percent CFSElow/percent CFSEhigh).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Primary CD8 T cell responses to HIV Env expressed by VSV are reduced 2- to 3-fold in the absence of CD4 T cells

Our goal was to determine whether responses to a vaccine Ag (HIV Env protein) expressed in a VSV-based vaccine vector as well as responses to a vector Ag (VSV N protein) could be generated and maintained in the absence of CD4 T cells. Although the immunodominant epitopes of HIV Env have not been identified for C57BL/6 mice, the response to the HIV Env epitope p18 (N-RGPGRAFVTI-C) that binds MHC class I H-2Dd in BALB/c mice has been extensively characterized (27), and an immunodominant epitope for VSV N (designated N2) was recently defined for BALB/c mice (N-MPYLIDFGL-C; D. Cooper, Wyeth Pharmaceuticals, unpublished observation). This N epitope binds the MHC class I H-2Ld molecule in BALB/c mice. Because CD4 T cell-deficient mice are not available on the BALB/c background, we backcrossed C57BL/6 CD4–/– mice onto the BALB/c background to generate BALB/c CD4–/–.

To analyze the primary responses to HIV and VSV N simultaneously, we immunized CD4+/+ and CD4–/– BALB/c mice with a live VSV recombinant (VSV EnvG) expressing an HIV Env protein in which the Env cytoplasmic domain was replaced with the VSV G cytoplasmic domain (28). We then assayed CD8 T cell responses to the VSV N2 epitope using an MHC class I H-2Ld pentamer bound to N2 or a class I H-2Dd tetramer bound to Env p18. BALB/c mice immunized with VSV EnvG made robust primary anti-HIV Env CD8 T cell responses at 8 days postimmunization in both the spleen and the lung (Fig. 1, A, C, E, and F) and these responses were reduced significantly (2- to 3-fold) in mice lacking CD4 T cells (Fig. 1, B and D–F). We also asked whether the CD8 T cells produced in the absence of CD4 help were able to kill target cells loaded with the Env p18 peptide in vivo (Fig. 1G). CD4-intact and CD4-deficient mice immunized with VSV EnvG were both able to kill HIV Env-loaded target cells (84.3 ± 1.2% and 84.9 ± 3.1% specific killing, respectively) and there was no significant difference between the two groups (p = 0.83). These results demonstrate clearly that a functional CTL response to HIV Env is generated independent of CD4 T cell help.


Figure 1
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  		FIGURE 1. Primary responses to HIV Env are reduced in CD4-deficient mice. Female BALB/c mice were immunized i.m. with 5 x 105 PFU of rVSV expressing HIV Env (VSV EnvG). Primary anti-HIV Env CD8 T cell responses were assayed in the spleen (A, B, and E) and lung (C, D, and F) of CD4 T cell-intact (A, C, E, and F) and CD4 T cell-deficient (B, D, E, and F) mice 7 days after primary immunization. Ag-specific CD8 T cells were defined as those expressing low levels of CD62L and binding HIV Env tetramer (Tet+CD62Llow, upper left quadrant). Graphs (E and F) represent average percent of CD8 T cells that are CD62Llow and bind HIV Env tetramer ± SEM. Data are representative of three separate experiments in each of which there was a minimum four animals per group. Cytotoxicity against the HIV Env peptide was tested in vivo (G) at 8 days postimmunization. Graph represents average specific killing ± SEM where percent-specific killing was determined as a ratio between CFSEhigh (target) and CFSElow (nontarget) cells recovered from recipient mice. Data are representative of two separate experiments in which a total of 15 CD4+/+ and 7 CD4–/– mice were analyzed.

 
Primary responses to VSV N2 are intact in CD4-deficient mice

To determine whether CD8 T cell responses to a different Ag might have a different requirement for CD4 T cell help, we assayed responses to the VSV N2 epitope bound by the BALB/c allele H-2Ld in the samples from the same mice used to assay responses to Env above. Responses to the VSV N2 epitope were lower (in terms of the percentage of Ag-specific CD8 T cells generated) than those to the HIV Env p18 epitope, but were still substantial (Fig. 2). Interestingly, CD4-intact and CD4-deficient mice produced equivalent numbers of VSV N2-specific CD8 T cells in both the spleen and the lung after the primary immunization (Fig. 2) showing that this primary response is completely independent of CD4 T cell help.


Figure 2
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  		FIGURE 2. Primary responses to VSV N2 are reduced in CD4-deficient mice. Female BALB/c mice immunized i.m. with 5 x 105 PFU of rVSV-expressing HIV Env (VSV EnvG, Fig. 1) were also analyzed for the development of anti-VSV N2 CD8 T cell responses. Primary anti-VSV N2 CD8 T cell responses were assayed in the spleen (A, B, and E) and lung (C, D, and F) of CD4 T cell-intact (A, C, E, and F) and CD4 T cell-deficient (B, D, E, and F) mice 8 days after primary immunization. Ag-specific CD8 T cells were defined as those expressing low levels of CD62L and binding VSV N2 tetramer (Tet+ CD62Llow, upper left quadrant). Graphs (E and F) represent average percent of CD8 T cells binding VSV N2 tetramer ± SEM. Data are representative of multiple experiments in which there was a minimum of four animals per group.

 
Memory and recall responses to HIV Env are partially CD4 independent

Several recent studies have shown that while CD4 T cells are often dispensable in the priming of CD8 T cell responses to live pathogens, they are normally required for long-term maintenance of these responses (13, 14). The signals provided by CD4 T cells for long-term maintenance of CD8 cells could be Ag specific or nonspecific and have not been defined.

To examine the requirement for CD4 cells in the long-term CD8 T cell responses to Env, we assayed memory and recall responses at 30, 60, or 90 days after primary immunization of BALB/c CD4+/+ or CD4–/– mice with VSV EnvG. Resting (memory) T cell levels to HIV Env p18 and VSV N2 were assayed simultaneously at each time point by MHC class I tetramer staining and included both CD62Lhigh and CD62Llow cells. To test the ability of memory cells to re-expand in these animals, parallel groups of immunized mice were boosted at 30, 60, or 90 days with a vaccinia virus expressing HIV Env (vPE16; Ref. 29). These animals were sacrificed 7 days after boosting (37, 67, or 97 days) and recall T cell responses analyzed. Any expansion of Ag-specific T cells after the boost must be a recall of memory T cells if its magnitude is greater than that seen in naive animals given the vaccinia-boosting vectors only. We are therefore defining such expansion as a memory-recall response, even though in some cases in CD4–/– animals it is no greater than the primary response to the VSV vector. Note that the primary responses to vaccinia vectors are much lower than the primary responses to VSV vectors.

We observed that memory-recall responses to HIV Env were preserved over the entire period in both CD4+/+ and CD4–/– mice (Fig. 3, A–D). HIV Env-specific T cells were detected in the spleens of unboosted CD4+/+ and CD4–/– animals at 30, 60, and 90 days after primary immunization (Fig. 3, A and B, Table I). However, note that the primary responses, memory cell levels, and the levels obtained after boosting were lower in the CD4–/– than in the CD4+/+ mice. In the spleens of CD4 +/+ mice, the boost generated responses that were 2- to 3-fold greater than those generated during the primary response (compare days 6–8 to days 37, 67, and 97 in Fig. 3A and Table I), while in the CD4–/– mice the responses after boosting were generally no greater than the primary response to the VSV vector (Fig. 3B). In the lung, the memory cell levels were below background in both the CD4–/– and CD4+/+ mice at the 60 and 90 day time points before boosting. However, after boosting, a vigorous recall response was observed 7 days later in both the spleen and the lung of CD4+/+ and CD4–/– animals. The difference between the lung recall response in the CD4+/+ and CD4–/– mice was <2-fold. This result shows that even though the memory cells may have declined to undetectable levels before boosting, they were still present and able to expand to high levels upon restimulation. In all cases, postboost responses (days 37, 67, and 97) were much greater than a primary response to the boosting vector and were therefore true memory recall responses (Tables I and II). For example, control naive CD4+/+ or CD4–/– mice primed with the vaccinia-Env virus generated 3.8 ± 0.4% or 0.7 ± 0.3% tetramer-positive cells, respectively (for HIV EnvP18), 7 days after infection while on day 67 (7 days postboost), CD4+/+ and CD4–/– mice generated 35.4 ± 1.7% and 5.3 ± 1.5% tetramer-positive cells, respectively.


Figure 3
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  		FIGURE 3. Long-term memory and recall responses to HIV Env are partially CD4 independent. Long-term memory CD8 T cell responses were assayed at 30, 60, and 90 days after primary immunization with VSV EnvG and before boosting. Additional mice were boosted at 30, 60, or 90 days after primary immunization with 1 x 105 PFU vaccinia virus expressing HIV Env protein (vPE16). Recall CD8 T cell responses were assayed at 7 days postboost (37, 67, or 97 days after primary immunization). Anti-HIV Env CD8 T cell responses were analyzed in the spleen and lung of immunized mice by tetramer and averages are graphed in A–D. Clear populations of HIV Env-specific CD8 T cells were identified in the spleens of both CD4 T cell intact and deficient mice at all time points tested. After 30 days postimmunization, Ag-specific cells could no longer be detected in the lung of either CD4+/+ or CD4–/– mice without boosting (C and D). In vivo CTL assays (E) were done at 7 days after primary immunization and at 7 days postboost with boosts at 30, 60, and 90 days. Data are representative of two to three separate experiments (days 6–37) in which a minimum of four animals per group were analyzed. Later time points represent a single experiment in which a minimum of four animals per group were analyzed. Average HIV-Env-specific killing in vivo ± SEM is graphed in E. A minimum of four CD4+/+ and four CD4–/– animals were analyzed at each time point.

 

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  		Table I. Primary, memory, and recall T cell responses to epitopes

 

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  		Table II. Primary responses in naive animals given vaccinia-boosting vectors

 
As in the primary response, when CTL function against HIV Env p18 was tested in vivo at 56, 67, or 97 days postpriming (7 days postboost in each case) both CD4+/+ and CD4–/– mice retained the ability to eliminate p18 peptide loaded cells in vivo (Fig. 3E). CD4–/– mice had significantly lower levels of cytotoxicity in vivo (p < 0.04, p < 0.04, p < 0.03 at days 56, 67, and 97) but it is unknown, because the level of killing still approached 100% in the 4-h assay, whether this difference would translate into a functional immunodeficiency in vivo. Together, these data demonstrate that the maintenance and function of significant numbers of memory CD8 T cells specific for HIV Env p18 does not require the presence of CD4 T cells for at least 3 mo after priming.

Memory and recall responses to VSV N2 are almost completely dependent on CD4 T cells

To determine whether the long-term memory and recall CD8 T cell responses to VSV N2 were CD4 dependent or independent, we used a vaccinia virus expressing VSV N (v38; Ref. 30) to boost an additional cohort of mice primed with VSV EnvG. As above, we then assayed resting and recall CD8 T cell responses at 30, 60, and 90 days postprime.

N2-specific memory cells were present in low but detectable numbers in the spleens of CD4+/+ mice at 30, 60, and 90 days, and also in CD4–/– mice at 30 and 60 days (Fig. 4, A and B, and Table I). VSV N2-specific cells were not detectable in the lungs of any unboosted mice at 30, 60, or 90 days postprime (Fig. 4, C and D, and Table I).


Figure 4
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  		FIGURE 4. Long-term memory and recall responses to VSV N2 are absent in CD4-deficient mice. Long-term memory CD8 T cell responses were assayed at 30, 60, and 90 days after primary immunization with VSV EnvG and before boosting. Additional mice were boosted at 30, 60, or 90 days after primary immunization with 1 x 105 PFU vaccinia virus expressing VSV N protein (v38). Recall CD8 T cell responses were assayed at 7 days postboost (37, 67, or 97 days after primary immunization). Anti-VSV N2 CD8 T cell responses were analyzed in the spleen and lung of immunized mice by tetramer and averages ± SEM are graphed in A–D. Data are representative of one to two separate experiments (days 6–37) in which a minimum of four animals per group were analyzed. Later time points represent a single experiment in which a minimum of four animals per group were analyzed.

 
When CD4+/+ mice were boosted at 30, 60, or 90 days, vigorous recall responses occurred in the spleen and lung. In contrast to the primary anti-VSV N2 response, in which CD4+/+ and CD4–/– responses were nearly equivalent, the recall response to N2 was absent in the spleens of CD4–/– mice by day 60. For example, at day 67 after a boost at day 60, the recall response was 50-fold below that seen in CD4+/+ mice (Fig. 4, Table I), and was not a true recall as it was equivalent to that seen in naive CD4–/– mice primed with the boosting vector (Tables I and II). Similarly, the postboost expansion of N2-specific cells in the lungs of CD4–/– mice was strongly reduced at day 67 (9-fold) compared with the responses seen in CD4+/+ mice. This contrasts with a <2-fold difference between the CD4+/+ and CD4–/– postboost responses to HIV Env encoded by the same virus at the same time point. In the lungs of CD4–/– mice, however, the day 67 and 97 recall responses to the N2 epitope did appear significantly greater than the primary response to the boosting vector in naive mice, indicating incomplete loss of N2 memory cells in this compartment (Tables I and II).

In summary, CD8 T cells specific for VSV N2 or HIV Env generated in the absence of CD4 are either lost or are less able to expand upon restimulation than those primed in CD4-intact animals. This defect is partially epitope-specific, with VSV N2-specific T cells losing the capacity to expand earlier and more completely than HIV Env-specific T cells.

Primary CD8 T cell responses to VSV N1 are largely independent of CD4 help in CD4–/– mice

The above results indicate that the capacity for re-expansion after secondary exposure to Ag is maintained or lost in an Ag- or epitope-specific manner. Although the recall response to VSV N2 epitope has not been directly assayed previously, the primary response to the VSV N epitope (N-RGYVYQGL-C; here designated VSV N1) (31, 32) has been characterized in C57BL/6 mice. An early study using different methods showed a strong requirement for CD4 T cell help in the primary CTL response to VSV (23), but a more recent study using a tetramer specific for VSV N1 found that anti-VSV N1 CD8 T cells were generated in nearly equal numbers in intact and CD4 T cell-deficient mice (25). This more recent study showed further that responses to a foreign Ag (OVA) initially presented by VSV could be boosted normally in the absence of CD4 help.

Because our results with the VSV N2 epitope contrasted with this previous work, we also analyzed the response to VSV N1 epitope in our system. We immunized mice with rVSV and quantified the CD8 T cell response to the VSV N1 epitope using an MHC class I tetramer (Fig. 5, A and B). When CD8 T cells were assayed at 8 days after primary immunization, there were 2- to 3-fold fewer VSV N1-specific T cells in the spleen of CD4–/– mice than in spleen of CD4-intact animals (Fig. 5A). A smaller difference was seen in the lung (Fig. 5B). Our results are thus consistent with those of Marzo et al. (25) who used CD4 cell depletion by Ab rather than CD4–/– mice, but also measured responses to the N1 epitope.


Figure 5
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  		FIGURE 5. Primary CD8 T cell responses to VSV N1 are reduced in CD4-deficient mice. Female C57BL/6 mice were immunized i.m. with 5 x 105 PFU rVSV. Primary anti-VSV CD8 T cell responses to the VSV N1 epitope were assayed in the spleen (A) and lung (B) of CD4 T cell-intact and CD4 T cell-deficient mice 8 days after immunization. Ag-specific CD8 T cells were defined as those expressing low levels of CD62L and binding VSV N1 tetramer. Graphs represent average percent of CD8 T cells binding VSV N1 tetramer ± SEM. Data are representative of two separate experiments in which a total of 11 animals per group were analyzed. Cytotoxicity against the VSV N1 peptide was tested in vivo (C) at 8 days postimmunization. Graph represents average specific killing ± SEM where percent-specific killing was determined as a ratio between CFSEhigh (target) and CFSElow (nontarget) cells recovered from recipient mice (n = 3 for both groups). A separate cohort of MHC class II–/– mice (n = 4) was immunized with rVSV as above. Levels of VSV N1 tetramer-specific T cells generated in these animals were not significantly different from those generated in CD4–/– mice (n = 11) (D).

 
To determine whether the reduction in the number of VSV N1-specific CD8 T cells caused a functional immunodeficiency in the CD4–/– mice, we performed a cytotoxic T cell assay in vivo. In this assay, killing of target cells loaded with VSV N1 peptide was very efficient and not significantly different in immunized CD4+/+ and CD4–/– mice (Fig. 5C). This experiment demonstrates that the VSV N1-specific CD8 T cells generated in the absence of CD4 T cell help are functional in vivo. Our results support the conclusion that primary CD8 T cell responses to the VSV N1 epitope are largely independent of CD4 T cell help.

MHC class II–/– mice also show no significant defect in primary response to the VSV N1 epitope

Recent studies have demonstrated that T cell development and positive selection can be abnormal in CD4–/– mice (33). These mice exhibit an abnormal population of cells that express the CD8 coreceptor molecule but are MHC class II restricted. These aberrant CD8 T cells can negatively affect the generation of normal CD8 T cell responses to a variety of stimuli (33). Because such cells might influence our results, we repeated the analysis of CD8 T cell responses in MHC class II–/– mice (26, 34), in which CD4 T cells do not develop because they cannot be positively selected. There were slightly more anti-VSV N1 T cells generated in the spleen of MHC class II–/– mice than in CD4–/– mice (10.2 ± 2.0% vs 6.6 ± 0.9%, Fig. 5D) at 8 days after primary immunization but the difference was not statistically significant (p = 0.12). In the lung, MHC class II–/– mice had slightly lower responses than CD4–/– mice (26.9 ± 6.6% vs 34.6 ± 3.6%, Fig. 5D). This difference was also not statistically significant (p = 0.63). These data confirm that CD4–/– mice are suitable for analyzing the requirement for CD4 cells in generation of CD8 T cells. Apparently, the class II-restricted CD8 T cells generated in these animals do not negatively affect the generation of Ag-specific CD8 T cells.

Memory and recall responses to VSV N1 are independent of CD4 help

To determine the role of CD4 T cell help in maintaining short-term recall CD8 T cell responses to VSV N1 in our system, we assayed anti-VSV N1 responses at 30 days after primary immunization. We also boosted animals 30 days after primary immunization with a vaccinia virus expressing the VSV N protein (v38) and assayed recall responses to VSV N1 at 7 days after the boost. At 30 days after primary immunization a clear population of resting CD8 T cells (defined as CD62Lhigh or CD62Llow and VSV N1 tetramer+) was present in the spleen of both CD4-intact (1.3 ± 0.1%) and CD4-deficient (0.33 ± 0.03%) mice (Fig. 6A). Tetramer-positive cells were also present in the lungs of both CD4+/+ (8.5 ± 1.9%) and CD4–/– (2.3 ± 0.3%) mice at this time (Fig. 6B). After boosting, the VSV N1-specific T cells expanded up to 30-fold (Fig. 6A) in both CD4+/+ and CD4–/– mice. There were no significant differences in the numbers of VSV N1-specific cells in either the spleens or the lungs of CD4-intact vs CD4-deficient animals. Similar results were obtained in MHC class II–/– mice immunized and boosted on the same schedule (data not shown). Although we have not followed these responses for as long a time as the responses to the N2 or HIV Env epitopes, these data demonstrate that CD8 T cell responses to VSV N1 can be maintained and recalled in the absence of CD4 T cells.


Figure 6
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  		FIGURE 6. Resting and memory T cell responses to VSV N1 are intact in CD4-deficient mice. Resting CD8 T cell responses were assayed at 30 days after primary immunization and before boosting. Mice were boosted at 30 days after primary immunization with 1 x 105 PFU vaccinia virus expressing VSV N protein (v38) (n = 10/group). Recall CD8 T cell responses were assayed at 7 days postboost. Graphs represent average percent of CD8 T cells binding VSV N1 tetramer ± SEM in the spleen (A) or lung (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We undertook this study to determine whether the CD8 T cell response to immunization with rVSV vectors is dependent upon CD4 T cell help. This issue is of particular importance if rVSV vaccines are to be administered to immunocompromised individuals. We observed that some, but not all, CD8 T cell responses to Ags expressed by rVSV are CD4 dependent. Primary responses to rVSV itself, and to foreign Ags expressed by rVSV, were independent of CD4 help. This is consistent with the results of Marzo et al. (25). In contrast, recall responses to Ags expressed by rVSV are CD4 dependent to different degrees, depending on the Ag. Specifically, when CD4-deficient mice were immunized with a rVSV expressing HIV Env, CD8 T cells specific for HIV Env P18 could be recalled and re-expanded for up to 3 mo postimmunization, while those specific for the VSV N2 epitope could not.

There are several possible explanations to account for the difference in recall potential between HIV Env- and VSV N2-specific T cells. Because HIV Env P18 is a dominant epitope, it is possible that "immunodomination" skewed the immune response toward Env and away from N. Related factors, such as the affinity of an epitope for the MHC class I molecule on which it is presented, and competition for class I molecules could also have influenced the outcome. However, because HIV Env P18 and VSV N2 are presented on different MHC class I molecules (Dd and Ld, respectively), it seems unlikely that either of these factors account for the difference. Also, the primary VSV N2 response is the same in animals infected with VSV lacking the Env gene. Another potential explanation is that relative abundance of the two proteins influenced the primary expansion, and subsequent maintenance, of CD8 T cells. However, expression from the VSV genome is polar, with the protein encoded by the first gene (VSV N) expressed at much higher levels than the protein encoded by the fifth gene (HIV Env).

Finally, it would be expected that the lower frequency of anti-VSV N2 cells generated after the primary response would result in retention of a smaller number of memory cells for VSV N2 vs Env p18. This phenomenon was recently described in detail by La Gruta et al. (35). Although memory precursor frequency may be a contributing factor, it would not explain the complete failure of N2-specific cells to re-expand in CD4-deficient animals. At days 8, 30, and 60, the frequency of VSV N2-specific cells in the CD4+/+ and CD4–/– mice was similar in the spleen. However, on days 60 and 90 cells in the CD4+/+ but not the CD4–/– mice re-expanded after boosting. Furthermore, HIV Env-specific cells reached undetectable levels in the spleen of CD4–/– mice at 60 and 90 days postprime, yet still re-expanded to high levels upon boosting. We propose that the requirement for CD4 help in the maintenance of CD8 T cells may be Ag specific due to some ongoing requirement for cognate interaction (either direct or via an APC intermediate) between CD8 T cells and CD4 T cells. Our data are consistent with the following model. After priming with a rVSV vaccine, the number of CD8 T cells generated varies according to the immunodominance of the epitope (Env P18 > N2). These primary responses are effectively independent of CD4 T cell help for both epitopes. After contraction of the primary response, cells enter the CD8 memory pool in numbers proportional to those generated during priming (Env P18 > N2). Thereafter (2–3 mo), a cognate interaction maintains the capacity of the memory CD8 T cells to re-expand in the CD4+/+ mice. This interaction is not maintained in the CD4–/– mice and is differentially lost depending on the epitope (N2 quicker than Env P18).

Although the mechanism is not yet clear, our results indicate that there are epitope-specific requirements for CD4 help in the maintenance of memory CD8 T cell responses. Further investigation of this phenomenon could be valuable in the ongoing effort to design Ags for an AIDS vaccine that could protect against disease in the context of reduced or declining CD4 T cell help.


   Acknowledgments
 
We thank Dr. David Cooper (Wyeth Pharmaceuticals, Pearl River, NY) for providing the sequence of the N2 epitope and for the N2 pentamer. We also thank Dr. Leo LeFrancois (University of Connecticut Health Cener, Farmington, CT) for the N1 tetramer. MHC class I Dd tetramer-binding HIV Env p18 was provided by the National Institutes of Health Tetramer Facility.


   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 Grants R37-AI40357 and R0-AI45510 and by a HIV-1 Vaccine Design and Development Team contract (NIH N01-AI-25458). Back

2 Address correspondence and reprint requests to Dr. John K. Rose, Department of Pathology, Yale University School of Medicine, 310 Cedar Street (LH315), New Haven, CT 06510. E-mail address: john.rose{at}yale.edu Back

3 Abbreviations used in this paper: VSV, vesicular stomatitis virus; VSV N, VSV nucleocapsid; RT, room temperature. Back

Received for publication November 1, 2006. Accepted for publication March 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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