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The Journal of Immunology, 1999, 163: 861-867.
Copyright © 1999 by The American Association of Immunologists

The Antiviral Activity of HIV-Specific CD8+ CTL Clones Is Limited by Elimination Due to Encounter with HIV-Infected Targets1

Denise M. McKinney*, Deborah A. Lewinsohn{dagger}, Stanley R. Riddell{dagger}, Philip D. Greenberg{dagger} and Donald E. Mosier2,*

* Department of Immunology, Scripps Research Institute, La Jolla, CA 92037; and {dagger} Program in Immunology, Fred Hutchinson Cancer Research Center, and Departments of Medicine and Immunology, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adoptive immunotherapy of virus infection with viral-specific CTL has shown promise in animal models and human virus infections and is being evaluated as a therapy for established HIV-1 infection. Defining the individual obstacles for success is difficult in human trials. We have therefore examined the localization, persistence, and antiviral activity of HIV-1 gag-specific CTL clones in both HIV-1-infected and uninfected haplotype-matched human (hu)-PBL-SCID mice. Injection of gag-specific clones but not control CTL into HIV-1-infected hosts reduced plasma viremia by >10-fold but failed to eliminate the virus infection from most treated animals. The failure to eradicate virus did not reflect selection of escape variants because the gag epitope remained unmutated in virus isolates obtained after CTL therapy. Injection of carboxyfluorescein diacetate succinimide ester-labeled CTL demonstrated markedly different fates for gag-specific CTL in the presence or absence of HIV-1 infection. HIV-1-specific CTL rapidly disappeared in infected recipients, whereas they were maintained at high numbers in uninfected mice. By contrast, control CTL were long lived in both infected and uninfected recipients. Thus, interaction of CTL with virus-infected target cells in vivo leads not only to target destruction but also to the rapid disappearance of the infused CTL, and it limits the capacity of CTL therapy to eliminate HIV-1 infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Primary HIV-1 infection generally elicits a potent anti-viral CTL response (1, 2, 3, 4), which contributes to the containment of viral replication but rarely results in eradication of the virus (5). Therefore, approaches to augment the magnitude of the CTL response, such as vaccination or adoptive T cell transfer, are being explored in the clinic. However, HIV-1 has evolved multiple mechanisms to evade the CTL response. The high mutation rate associated with error-prone virus replication can lead to the rapid generation of viruses with variant epitopes no longer recognized by CTL (6, 7, 8, 9, 10). Additionally, expression of the viral accessory protein Nef down-regulates class I MHC expression on infected cells (11), and may up-regulate Fas ligand (12) or soluble TNF-{alpha} (13), leading to reduced efficiency of CTL recognition and potential killing of CTL effectors on interaction with infected target cells. Indeed, the primary CTL response to HIV-1 infection is often oligoclonal by TCR analysis (14, 15), and some of the CTL clones that mediate this response rapidly disappear early in the course of disease (16). It is not clear whether this disappearance reflects depletion of CTL during the active effector stage or the normal elimination by apoptosis of CTL the Ag of which has been eliminated, or, in this case, may have been altered by mutation (8).

There is considerable evidence that CTL contribute not only to the control of the burst of viremia after primary infection (17) but also continue to control HIV-1 throughout the course of infection (18, 19). A potent CTL response is a predictor of lower viral load and better survival (20, 21, 22). Nonetheless, CTL activity eventually diminishes, and many CD8+ T cells show evidence of clonal exhaustion (23), virus production increases, and patients progress to AIDS. Efforts to augment the CTL response by the adoptive transfer of CTL clones have met with limited success (24, 25, 26). Recently, we have demonstrated that such CTL therapy has antiviral activity but that this effect is only transient (26). To elucidate the obstacles to establishing a more effective CTL response to HIV-1, we have performed parallel studies in HIV-1-infected patients (25, 27) and studies of CTL transfer in HIV-1-infected human (hu)-PBL-SCID mice3 (28, 29). We report here that HIV-1 gag-specific CTL clearly mediate an antiviral effect in vivo, as reflected by a significant reduction in plasma virus load in infected hu-PBL-SCID mice, but, similar to the observations in treated humans (26), that the antiviral activity is transient. This transient activity was not due to the generation of epitope escape mutants but rather to the rapid disappearance of the CTL as a consequence of target recognition. The implication of these results for strategies to promote the immunologic control of HIV infection are discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human donors

All cells used in these experiments were derived from one pair of monozygotic twins discordant for HIV-1 infection. CTL clones were generated from the infected twin as described below. PBMC were prepared as previously described (30, 31) after leukapheresis of the uninfected twin and frozen in vapor-phase liquid N2 for up to 1 year before use. The CCR5 genotype of the twins was homozygous wild type for the 32-bp deletion allele (32).

Reconstitution of SCID mice with human PBL

C.B-17 SCID mice were bred under specific-pathogen-free conditions at Scripps Institute (La Jolla, CA) and tested for mouse IgM production at 8 wk of age. Mice with <5 µg/ml of IgM were engrafted with PBMC prepared from the uninfected twin donor. SCID mice were injected with 25–30 x 106 PBMC i.p. and checked for plasma levels of human IgG after 12–13 days. Mice with >100 µg/ml human IgG were used for HIV-1 infection.

Generation of human CTL clones

The methods for the generation of HIV gag-specific and control clones have been described (25). Briefly, gag-specific CTL bulk cultures were generated by culturing T cells derived from PBMC of a single HIV-infected individual with vaccinia-gag recombinant virus-infected, UV-irradiated autologous macrophages as stimulators. CD8+ gag-specific CTL clones were isolated by limiting dilution cloning, using as responders CD4-depleted T cells from day 14 bulk cultures and anti-CD3 mAb ( 30 ng/ml, Zymed, San Francisco, CA) + IL-2 at 25 U/ml for TCR stimulation. The gag-specific CTL clone used for these experiments was HLA-B27-restricted and recognized the gag p17 matrix protein position 19–27 peptide IRLRPGGKK (D. A. Lewinsohn, P. D. Greenberg, and S. R. Riddell, manuscript in preparation). This epitope is highly conserved among HIV-1 isolates, and the consensus clade-B sequence is shared by the HIV-1 isolates SF2 and JR-CSF used in these experiments (HIV Molecular Immunology Database, Los Alamos National Laboratory, Los Alamos, NM; http://hiv-web.lanl.gov/immunology/index.html). The control CTL population was similarly cloned by limiting dilution. Its specificity is unknown, but it does not react to HIV or HIV-derived peptides.

Rapid expansion of human CTL clones

gag-specific or control CTL cells (2–4 x 105/flask) were cultured with 25 x 106 allogeneic {gamma}-irradiated (4000 cGy) PBMC, 5 x 106 allogeneic, {gamma}-irradiated (8000 cGy) EBV-transformed lymphoblastoid cells, and 30 ng/ml anti-CD3 mAb (Zymed, San Francisco, CA) in RPMI supplemented with 10% human serum (CTL media). After 24 h, 50 U/ml recombinant human IL-2 (rh-IL-2) was added to each flask. On day 5 of stimulation, the anti-CD3 mAb was removed, and the CTL were resuspended in CTL media with 30 U/ml rh-IL-2. Cultures were supplemented with an additional 30 U/ml rh-IL-2 on days 7 and 9 of the stimulation. On day 12, the cells were washed, and 1 x 106 CTL/well were plated into 24-well tissue culture plates in 1–2 ml CTL media with 2 x 106 allogeneic {gamma}-irradiated PBL to induce resting CTL. After 4 days, cells were injected into SCID mice reconstituted with PBL from the uninfected monozygotic twin.

CFSE labeling of CTL and flow cytometry analysis

CTL were labeled with carboxyfluorescein diacetate succinimide ester (CFSE, Molecular Probes, Eugene, OR) by incubation at 37°C at 1 µM in RPMI 1640 + 10% FBS for 10 min (33, 34). The cells were washed three times before injection into hu-PBL-SCID mice or analysis by flow cytometry for baseline fluorescence intensity. Cells recovered from the peritoneal cavity or regional lymph nodes (LN) of hu-PBL-SCID mice were stained with PE-labeled Abs to human CD3, CD4, CD8, or CD45 and mouse H-2Kd (PharMingen, San Diego, CA) and analyzed on a FACScan (Becton Dickinson, Mountain View, CA) flow cytometer. Adequate discrimination of CFSE-positive CD8 T cells was confirmed using allophycocyanin-coupled anti-CD8 Abs (Becton Dickinson) and a dual laser FACSCalibur instrument (Becton Dickinson). A minimum of 104 cells were analyzed using Cellquest software (Becton Dickinson). The intensity of CFSE staining per cell did not decrease during the time course of these experiments (Fig. 4Go), indicating that little or no cell division of the injected CTL was occurring.



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  		FIGURE 4. Fluorescence intensity of CFSE labeling in CD8+ cells recovered at days (d.) 0 and 2 in the experiment shown in Fig. 3GoB, i.e., in the presence of HIV-1 infection. The median channel of fluorescence intensity (FL1) CFSE staining is shown in each panel. The scale of cell counts differs in each panel, primarily because of the lower recovery of gag-specific CTL in the day 2 sample.

 
Virus infection

Two strains of HIV-1, SF2 (a T cell line-tropic, CXCR4-using isolate) and JR-CSF (a macrophage-tropic, CCR5-using isolate), were used in these experiments. SF2 was provided by Jay Levy, and JR-CSF was obtained from the AIDS Reagent Repository, Rockville, MD. Virus stocks were produced in human PBMC that were activated by 2 days of exposure to 5 µg/ml PHA followed by an additional 2 days of culture in 25 U/ml human IL-2. The tissue culture infectious dose (TCID50) of the virus was established by limiting dilution, and animals infected with 103 TCID50 per mouse by i.p. injection of virus. Both SF2 and JR-CSF retain the conserved epitope recognized by the gag-specific CTL. Although CTL administration reduced viral loads in SF2-infected hu-PBL-SCID mice (data not shown), the experiments presented below utilize JR-CSF-infected mice because the longer and more stable course of infection with this virus (30) made the determination of antiviral efficacy clearer.

Analysis of plasma viral load

Infection of hu-PBL-SCID mice with HIV-1 was determined by plasma HIV-1 RNA levels measured by the quantitative Roche PCR assay (Amplicor HIV Monitor, Roche Molecular Systems, Somerville, NJ). The limit of detection was 200–400 copies/ml depending on the plasma volume available.

Sequencing of recovered virus

HIV-infected and CTL-treated hu-PBL-SCID mice were sacrificed, and cells recovered from the spleen (SP), periportal LN, and peritoneal lavage (PW) were cocultured with PBMC for 1 wk. RNA was then extracted from the culture supernatant (Trizol method), and cDNA was made, amplified using Pfu DNA polymerase (Stratagene, La Jolla, CA) to minimize PCR errors with p17-gag-specific primers (5'-ATGGGTGCGAGAGCGTCA; 3'-CTTCTGATGATTCTAACAGGCCAGG) to yield a 294-bp product flanking the identified epitope, and cloned into the TOPO-TA cloning vector (InVitrogen, San Diego, CA). A minimum of six clones from the LN, PW, and/or SP were sequenced for each mouse using automated DNA sequencing and dye terminators (ABI 373, Scripps Core Sequencing Laboratory, La Jolla, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Impact of gag-specific CTL on established HIV-1 infection

Hu-PBL-SCID mice were generated using PBMC grafts from the uninfected monozygotic twin and infected with HIV-1 strain JR-CSF 2 wk later. After 7–9 days, 107 gag-specific or control CTL derived from the infected twin were injected i.p. into each animal. Plasma viral RNA levels were measured just before CTL injection and at 1, 2, and 7 days after injection. Selected mice, including all mice with plasma virus levels <2–400 copies/ml on day 7, were injected with 107 additional autologous PBMC on day 14 to provide additional target cells for virus infection, and plasma viral RNA levels were measured once more on days 21–24 after CTL injection. Virus was considered to have been eradicated only when viral RNA levels were undetectable at this last sample time, and virus could not be isolated by coculture.

Two replicate experiments (of 5 performed) using this protocol are shown in Figs. 1Go and 2. In each experiment, 5 hu-PBL-SCID mice per CTL treatment group were followed. In Fig. 1GoA it can be seen that viral RNA levels declined in 5 of 5 mice after injection of gag-specific CTL, whereas only 1 of 5 mice injected with control CTL showed a decline in viral RNA (Fig. 1GoB). Geometric mean viral RNA in the gag-specific CTL-treated group declined from 44,044 copies/ml before treatment to 2,209 copies/ml 2 days after treatment, a 20-fold decline (Table IGo, Expt. 107). Mean viral RNA levels in mice receiving equal numbers of control CTL increased from 10,256 copies/ml at baseline to 12,947 copies/ml 2 days after treatment, consistent with the progression of infection in untreated mice. By 7 days after CTL treatment, three mice (two mice receiving gag-specific CTL and one mouse receiving control CTL) had undetectable viral RNA levels in plasma (<200 copies/ml). To determine whether these mice were virus free, 107 autologous PBMC were injected into all mice to provide additional target cells for virus infection. One of the two mice receiving gag-specific CTL showed a rebound in viral RNA levels (Fig. 1GoA), but the other two mice still had undetectable levels of viral RNA. Parallel control groups receiving no CTL therapy maintained viral RNA levels in the detectable range throughout the time course of these experiments, so the occasional drop in viral load in a mouse receiving control CTL may be related to antiviral activity mediated in the absence of HLA-restricted killing, an effect previously observed with higher numbers of CTL in this model (28).



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  		FIGURE 1. Plasma viral RNA levels in individual hu-PBL-SCID mice infected with HIV-1 strain JR-CSF 7 days earlier and injected with 107 gag-specific CTL (A) or control CTL (B) on day 0. Bold line and filled circles, mean values for the five mice in each treatment group. On day 14, an additional 107 autologous PBMC were injected into any mice with undetectable viral RNA levels (<400 copies/ml in this experiment), and final viral RNA levels measured on day 21.

 

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  		Table I. Reduction in HIV-1 viral RNA by adoptive transfer of CTL1

 
A replicate experiment of nearly identical design is shown in Fig. 2Go. Injection of gag-specific CTL again resulted in a rapid fall in plasma viral RNA levels in 5 of 5 mice (Fig. 2GoA), with the mean value declining from 10,499 copies/ml on day 0 to 596 copies/ml on day 2 (Table IGo, Expt. 108). Injection of control CTL again failed to cause a reduction in viral load (Fig. 2GoB). By day 7 after CTL injection, 3 of 5 mice treated with gag-specific CTL had undetectable plasma viral RNA, but 2 of 5 mice treated with control CTL also showed undetectable viral RNA levels. On day 14 after CTL injection, additional autologous PBMC were injected into the mice with undetectable plasma viral RNA levels. Two of the 3 gag-specific CTL-treated mice showed an increase in plasma viral RNA at day 24 (Fig. 2GoA), and both of the control CTL-injected mice showed increased viral RNA (Fig. 2GoB). In both of these replicate experiments, mice with the highest starting viral RNA levels before CTL therapy showed the most rapid rebound after injection of gag-specific CTL.



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  		FIGURE 2. The conditions of this experiment are nearly identical with the one shown in Fig. 1Go, except that the detection limit for viral RNA was 200 copies/ml due to collection of more plasma at each sample point. Only mice with undetectable viral RNA levels were injected with additional PBMC on day 14, and the final measurement of viral RNA was made on day 24.

 
The short term impact of CTL therapy in all five replicate experiments is shown in Table IGo. Mean plasma viral RNA copy numbers for recipients of either control or gag-specific CTL are shown on the day of CTL injection and 2 days after CTL therapy. Mice injected with control CTL had a mean viral RNA increase of 0.370 log10 during this interval. In contrast, mice injected with gag-specific CTL had a mean viral RNA decrease of nearly -1 log and a median decrease of -1.25 log. In all five experiments, control groups of mice demonstrated increased viremia, and groups of mice receiving gag-specific CTL demonstrated decreased viremia. These results clearly show that injection of CTL into HIV-1-infected hu-PBL-SCID mice induced a transient reduction in viral RNA levels of significant magnitude but that complete clearance of virus was infrequent. In experiments 113 and 118, both gag-specific and control CTL were labeled with CFSE (see Materials and Methods) for tracking their fate (see below), and there is no indication from the data that CFSE labeling impaired their function in hu-PBL-SCID mice. Injection of gag-specific CTL resulted in a higher recovery of human CD4+ T cells in these experiments, but this trend was not statistically significant and would require larger number of animals to substantiate.

Sequencing of HIV-1 recovered from CTL-treated mice

The rebound in viral RNA levels after CTL treatment could have been due to the emergence of epitope escape mutants. We therefore PCR amplified p17 gag viral sequences from HIV-1 recovered from the peritoneal cavity, LN, or SP of CTL-treated hu-PBL-SCID mice and sequenced at least 6 clones from each mouse receiving gag-specific CTL and 5 clones from a mouse receiving control CTL. All 51 clones recovered had the identical coding sequence for the CTL epitope as the starting JR-CSF isolate (Table IIGo), although a few random silent base changes outside this region were seen. There was thus no indication that mutation of the p17 gag epitope contributed to the lack of effectiveness of CTL treatment.


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  		Table II. Sequences of p17 gag from JR-CSF isolates after gag-specific CTL or control CTL therapy

 
Tracking of CFSE-labeled CTL

To determine the in vivo fate of CTL injected into hu-PBL-SCID mice, both gag-specific and control CTL were labeled with CFSE before injection into SCID mice reconstituted with autologous PBMC. In two consecutive experiments, the labeled CTL were injected i.p. into either uninfected hu-PBL-SCID mice (Fig. 3GoA) or HIV-1-infected mice (Fig. 3GoB). Cells were recovered by PW and by harvesting local LN and the SP of mice at 1 h (day 0 in Fig. 3Go), and 1, 2, or 7 days after injection. CFSE-positive cells were counted by flow cytometry, using light scatter gating and staining with PE-conjugated anti-H-2Kd Ab to eliminate mouse cells from the analysis. In the absence of HIV-1 infection, both gag-specific and control CTL persisted in high numbers in the peritoneal cavity (Fig. 3GoA) and in lower numbers in the LN (data not shown). However, the survival of gag-specific CTL was markedly shortened in the presence of HIV-1 infection (Fig. 3GoB), whereas the survival of control CTL was not altered. This reduced recovery of CFSE-labeled CTL was not due to redistribution of CTL to other organ sites, because recovery of labeled CTL in infected but not uninfected mice showed a similar decline in SP and regional LN. In the experiment shown in Fig. 3Go, examination of SP cells on day 7 after CTL injection showed that 23% of CD8 cells were labeled in mice receiving control CTL vs 6% in mice receiving gag-specific CTL. Fewer labeled CTL were recovered in LN, but control mice had a proportionally higher recovery than mice injected with gag-specific CTL (2.5% vs 0.02%).



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  		FIGURE 3. Survival of CFSE-labeled CTL injected into uninfected hu-PBL-SCID mice (A) or HIV-1-infected mice (B). CTL were labeled as described in Materials and Methods, and 107 cells were injected i.p. into SCID mice reconstituted with autologous PBMC from the uninfected identical twin of the CTL donor. Two to three mice were sacrificed just after injection (day 0) and at 1, 2, and 7 days after CTL transfer, and labeled CD8+ cells were detected by two-color flow cytometry. The percentage labeled cells are expressed as a percentage of total CD8+ T cells, with the day 0 values ranging from 96 to 99%.

 
Fig. 4Go shows the CFSE staining intensity of CD8+ cells just after transfer to hu-PBL-SCID mice and at 2 days after transfer, and there is little change in the median channel of fluorescence intensity, indicating that little or no dilution of labeling due to cell division occurred. No change in CFSE labeling intensity per cell was observed during the 7-day duration of the experiment, demonstrating that the decrease in CFSE-labeled cells resulted from cell death rather than division. The cell loss was restricted to the CFSE-labeled CTL and did not include the unlabeled CD8 T cells contained in the original PBMC graft. For example, in the experiment shown in Fig. 3GoB, the fraction of CD8 T cells as a percentage of total human cells in the peritoneal cavity declined from 55 ± 12% on day 2 after injection of gag-specific CTL to 36 ± 2% on day 7, whereas the number of unlabeled CD8 T cells in the peritoneal cavity remained constant in the range between 4 and 8 x 105 during this period, consistent with the selective loss of CFSE-labeled cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results demonstrate that injection of gag-specific CTL into HIV-1-infected hu-PBL-SCID mice results in a substantial and significant reduction in viral load but that the effects of a single round of CTL treatment are transient and most mice remain infected with HIV-1. A major reason for the transient benefits of CTL treatment appears to be the selective and rapid loss of gag-specific CTL when they encounter HIV-1-infected target cells. Indeed, the alternative explanation for limiting the in vivo activity of the transferred CTL, the outgrowth of escape variants, appeared to play no role because we were unable to identify any mutations of the p17 19–27 gag epitope in viral isolates obtained from mice with persistent infection. This may in part reflect the specificity of the CTL clone selected, because the sequence of this epitope is highly conserved in multiple viral strains (35) and overlaps the nuclear localization signal of p17 matrix protein. Mutations in the KK residues at positions 26 and 27 of this protein (positions 8 and 9 of the epitope) have been shown to abolish interaction with a host protein and reduce virus infectivity (36); thus, mutations that alter this epitope might be lethal for the virus. A strong CTL response to primary infection (8) or adoptive CTL therapy of a patient with a Nef-specific CTL clone (24) has been shown to lead to mutation of the recognized epitope, suggesting that epitope mutations can represent an in vivo mechanism to evade CTL responses. However, our results suggest that in a setting that might select for diminished ability of the virus to escape by mutation, limited survival of CTL in vivo can be a formidable obstacle to viral elimination by the CTL.

Rapid disappearance of a clonal population of HIV-1-specific CD8+ T cells has been observed in primary infection of adult patients and in patients infused with CTL (16, 37), so the observations we have made are not unique to the hu-PBL-SCID animal model. Moreover, we have recently observed in a clinical trial in which HIV-1-infected patients were treated by the i.v. infusion of gene-marked HIV-1-specific CTL that the transferred CTL mediated a transient antiviral effect and that loss of antiviral activity was associated with disappearance of the CTL from the blood (26). However, it was not possible in that study to determine whether CTL disappearance from the blood reflected cell death, sequestration at peripheral sites, or nonspecific deletion in HIV-1 infection patients of any CTL regardless of Ag specificity. The present studies in the hu-PBL-SCID model extend these findings by providing the simultaneous controls of long term gag-specific CTL survival in uninfected hosts as well as long term survival of control CTL that are unable to recognize HIV-1 in the HIV-infected host. Thus, these studies suggest that shortened survival of HIV-specific CTL in HIV-infected individuals interferes with the ability of the CTL to control the infection.

The explanation for the short half-life of adoptively transferred or endogenously generated CTL will require further study. HIV-1-infected target cells may express death-inducing ligands such as Fas ligand or TNF-{alpha} and induce apoptosis in CTL (12, 13). Alternatively, high levels of Ag-specific CD4+ T helper activity may be required to sustain the CTL response in vivo, as has been observed in both murine and human settings of chronic virus infection (38, 39, 40). These helper cells are unlikely to survive primary infection in the hu-PBL-SCID model just as they fail to survive primary infection in most humans (40, 41). The relative contributions of each of these mechanisms for the observed relatively short half-life of adoptively transferred CTL are readily testable in the hu-PBL-SCID model.

The adoptive transfer of gag-specific CTL is much more effective at reducing viral load in hu-PBL-SCID mice than the transfer of 50 mg/kg of a potent neutralizing Ab (44). This leads us to believe that the generation of a potent CTL response by candidate vaccines or augmentation of the existing CTL response in infected individuals are appropriate primary goals of immunotherapeutic approaches to treat HIV-1 infection. Therefore, strategies that enhance the survival of CTL after Ag encounter, such as providing exogenous IL-2 (42) or genetically modified CD4 T cells resistant to HIV-1 (43), need to be pursued.


   Acknowledgments
 
We thank Richard Gulizia, Andrew Beernink, and Michael Neal for their skilled technical assistance; and the uninfected twin donor for his continued participation in this study.


   Footnotes
 
1 This work was supported by National Institutes of Health Strategic Program for Innovative Research in AIDS Therapy Grant AI36613. D.M.M. was supported by National Institutes of Health Training Grant T32 AI07244. This is Publication IMM-12039 from Scripps Research Institute. Back

2 Address correspondence and reprint requests to Dr. Donald E. Mosier, Department of Immunology-IMM7, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: Back

3 Abbreviations used in this paper: hu, human; CFSE, carboxyfluorescein diacetate succinimide ester; TCID50, 50% tissue culture infectious dose; gag, HIV-1 p17 matrix protein, a product of the HIV-1 gag gene; rh-IL-2, recombinant human IL-2; LN, lymph node(s); SP, spleen; PW, peritoneal lavage. Back

Received for publication January 22, 1999. Accepted for publication April 26, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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