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1 AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 15, Number 17, 1999, pp Mary Ann Liebert, Inc. Effect of Long-Term Infection with nef-defective Attenuated HIV Type 1 on CD4 1 and CD8 1 T Lymphocytes: Increased CD45RO 1 CD4 1 T Lymphocytes and Limited Activation of CD8 1 T Lymphocytes JOHN J. ZAUNDERS, 1 ANDREW F. GECZY, 2 WAYNE B. DYER, 2 LARISSA B. MCINTYRE, 3 MARGARET A. COOLEY, 4 LESLEY J. ASHTON, 5 CAMILLE H. RAYNES-GREENOW, 2 JENNY LEARMONT, 2 DAVID A. COOPER, 1,5 and JOHN S. SULLIVAN 2 ABSTRACT Members of the Sydney Blood Bank Cohort (SBBC) have been infected with an attenuated strain of HIV-1 with a natural nef/ltr mutation and have maintained relatively stable CD4 1 T lymphocyte counts for years. Flow cytometric analysis was used to examine the phenotype of CD4 1 and CD8 1 T lymphocytes in these subjects, including the immunologically important naive (CD45RA 1 CD62L 1 ), primed (CD45RO 1 ), and activated (CD38 1 HLA-DR 1 and CD28 2 ) subsets. The median values were compared between the SBBC and control groups, comprising age-, sex-, and transfusion-matched HIV-1-uninfected subjects; transfusion-acquired HIV-1-positive LTNPs; and sexually acquired HIV-1-positive LTNPs. Members of the SBBC not only had normal levels of naive CD4 1 and CD8 1 T lymphocytes, but had primed CD45RO 1 CD4 1 T lymphocytes at or above normal levels. Furthermore, these primed cells expressed markers suggesting recent exposure to specific antigen. SBBC members exhibited variable activation of CD8 1 T lymphocytes. In particular, SBBC members with undetectable plasma HIV-1 RNA had normal levels of activated CD8 1 T lymphocytes. Therefore, the result of long-term infection with natural nef/ltr mutant HIV-1 in these subjects suggests a decreased cytopathic effect of attenuated HIV-1 on susceptible activated CD4 1 T lymphocyte subsets in vivo, and minimal activation of CD8 1 T lymphocytes. INTRODUCTION HE SYD NEY BL OOD BA NK CO HORT (SBBC) recipients T 1 form a unique cluster of HIV-1-seropos itive subjects who were infected years ago through blood transfusion with an attenuated, nef/ltr-defective strain of HIV-1. 2 The amount of HIV-1 RNA and DNA in peripheral blood from these patients is particularly low, and their CD4 1 cell counts are either stable (four of six individuals studied long term) or declining slowly (two of six individuals). 3 Nef-defective HIV-1 has been discussed as a candidate vaccine in humans, 4 since vaccination with nef-defective SIV has been shown to be protective in macaques. 5 Strong HIV-specific cytotoxic T lymphocyte responses have been demonstrated in three of six recipients from the cohort. 6 However, the long-term effect of infection with attenuated HIV-1 on the human immune system requires careful analysis, since live attenuated SIV may cause immunodeficiency in macaques 7 and there is evidence of CD4 1 cell loss in some SBBC recipients. 3 We have shown that in vitro lymphoproliferation, an indicator of immune function, was normal in the SBBC, 8 but detailed study of CD4 1 and CD8 1 T lymphocyte subsets in these patients has not been undertaken. Attention has been focused on the levels of naive (CD45RA 1 CD62L 1 ) CD4 1 and CD8 1 T lymphocytes in HIV- 1 Centre for Immunology, St. Vincent s Hospital, Darlinghurst, NSW, 2010 Australia. 2 Australian Red Cross Blood Service-NSW, Sydney, NSW, 2000 Australia. 3 NSW Health Department, North Sydney, NSW, 2060 Australia. 4 Department of Microbiology and Immunology, University of NSW, Kensington, NSW, 2033 Australia. 5 National Centre in HIV Epidemiology and Clinical Research, Darlinghurst, NSW, 2010 Australia. 1519

2 1520 ZAUNDERS ET AL. 1 infection, since loss of these cells has been associated with progressive disease, 9,10 beginning during primary HIV-1 infection. 11 Only limited recovery of naive CD4 1 T lymphocytes is achieved with antiretroviral therapy It is therefore important to determine whether long-term infection with attenuated HIV-1 leads to an erosion of the number of naive CD4 1 or CD8 1 T lymphocytes. Also, a decrease in the proportion of CD26 1 CD4 1 T lymphocytes has been reported as being a characteristic of HIV-1-infected long-term nonprogressors (LT- NPs). 15 CD8 1 cytotoxic T lymphocytes (CTLs) may be involved in control of progression during the asymptomatic phase of HIV- 1 infection. 16,17 Human CD8 1 CTLs specific for HIV-1 express the activation markers CD38 and HLA-DR 18 and have reduced expression of CD28. 19,20 Paradoxically, elevated numbers and proportions of circulating CD8 1 CD38 1 and CD8 1 CD28 2 cells are consistently found to indicate a poor prognosis in HIV-1 infection Conversely, relatively lower levels of CD38 expression and higher levels of CD28 expression have been described in some studies of HIV-1-infected LTNPs. 15,21,25 27 We have therefore examined the cell surface phenotype of peripheral blood CD4 1 and CD8 1 T lymphocytes from six recipient members of the SBBC, and compared them with three control groups, including uninfected individuals matched for age, sex, and transfusion; LTNPs with transfusion-acquir ed HIV-1 infection; and LTNPs with sexually acquired HIV infection. Subjects MATERIALS AND METHODS Individuals were divided into four groups: 1. The SBBC: Comprising six LTNPs with a common donor. 1 Results for the present study are from samples collected from these patients over a 3-year period to July Table 1 describes demographic and longitudinal viral load and CD4 1 and CD8 1 cell counts for these subjects over the study period. Extensive clinical and laboratory results for these patients have been reported elsewhere Control HIV-1-uninfec ted cohort: Comprising 18 HIV-1- uninfected individuals, including 3 individuals matched for age TA BL E 1. and sex for each member of the SBBC; plus 17 HIV-1-uninfected individuals, including at least 2 individuals matched for age, sex, and time of transfusion. 3. Transfusion-acq uired long-term nonprogressors (TA-LT- NPs): Comprising 7 LTNPs infected with HIV-1 through receipt of transfusion products years ago from HIV-1-positive blood donors who have progressed to AIDS Sexually acquired long-term nonprogressors (SA-LTNPs): Comprising 14 LTNPs with sexually acquired HIV-1 infection, infected with HIV-1 for more than 11 years. 8 The study was approved by the Australian Red Cross Blood Service-New South Wales (ARCBS-NSW) Institutional Ethics Committee and informed consent was obtained from all participants. Monoclonal antibodies Monoclonal antibodies (MAbs) to the following cell surface proteins were used: CD3 PerCP, CD38 PE, HLA-DR FITC, CD8 FITC, CD25 PE, CD28 PE, CD62L (Leu-8) FITC, CD57 FITC, and CD95 PE (Becton Dickinson, San Jose, CA); CD4 ECD, CD8 ECD, CD45RA (2H4) RD1, CD11a (S6F1) RD1, CD26 (Ta1) RD1, and CD29 (4B4) FITC (Coulter Electronics, Hialeah, FL); CD45RO FITC and CD45RO PE (Dako, Carpinteria, CA); and Ki-67 FITC (Immunotech, Marseilles, France). All MAbs were used at the manufacturer-recomm ended concentrations. Flow cytometry MAbs were incubated with 100 m l of acid citrate dextrose (ACD)-anticoagul ated whole blood for min at 25 C before lysis with Optilyse C (Immunotech) for 10 min at 25 C, followed by one wash with phosphate-buffere d saline (PBS). Stained cells were resuspended in 0.5 ml of PBS containing 0.5% paraformaldehy de and analyzed within 24 hr of preparation. Samples were analyzed on a Coulter EPICS XL flow cytometer, with compensation levels checked daily. Lymphocyte gates were set manually on forward and side scatter and 10,000 lymphocytes were analyzed (Fig. 1A). CD3 PerCP and either CD4 ECD or CD8 ECD staining (Fig. 1B) were used to set gates for subsequent analysis of two-color FITC/PE histograms (Fig. 1C). Positively stained cells were determined by compar- HIV-1 VIR AL LO AD A ND LY M PHO CY TE SUBSETS IN IN DIVIDUA L ME M BE RS OF TH E SBBC C49 C135 C64 C98 C54 C18,,, m m Sex Female Male Female Male Male Male Age (years) Number of study visits a Plasma HIV-1 RNA b (copies/ ml) ( ) ( ) CD4 T lymphocytes (cells/ l) ( ) ( ) ( ) ( ) ( ) CD8 T lymphocytes (cells/ l) ( ) ( ) ( ) ( ) ( ) a C18 died at age 83 years, within the study period, from causes unrelated to HIV-1. 3 b Numbers represent the median (interquartile range) of observations for each subject.

3 CD4 1 AND CD8 1 T CELLS IN ATTENUATED HIV-1 INFECTION 1521 FIG. 1. Four-color flow cytometric analysis of CD45RA 1 and CD45RO 1 CD4 1 T lymphocytes: representative histograms for subject C49, showing gating strategy for four-color analysis of T lymphocyte subsets. Histogram A shows forward scatter/ side scatter gate to identify lymphocytes. Histogram B, gated on lymphocytes, shows CD3 PerCP versus CD4 ECD, with the region used to gate CD3 1 CD4 1 lymphocytes. Histogram C, gated on lymphocytes and CD3 1 CD4 1, cells, shows CD45RO PE fluorescence versus CD45RA PE. The upper left quadrant is used to quantify CD45RA 1 CD45RO 2 cells, while the lower right quadrant measures CD45RA 2 CD45RO 1 cells. ison with nonspecific staining controls. Cells identified as CD45RA 1 CD45RO 2 and CD45RA 2 CD45RO 1 are, respectively, referred to as CD45RA 1 and CD45RO 1 populations, while cells that were weakly positive for both markers were not included in the analysis (Fig. 1C). Intracellular staining for Ki-67 was performed on whole blood after cell surface staining, lysis, and fixation with FACS lysing solution (Becton Dickinson), and permeabilization with FACS permeabilizing solution (Becton Dickinson). Permeabilized cells were incubated with MAb and 5 m l of human gammaglobulin (kind gift from C.S.L., Melbourne, Australia) for 45 min at 25 C and washed once. Samples stained intracellularly were analyzed by gating on CD3 PerCP and side scatter with an FL4 threshold. Specificity of staining was demonstrated by blocking with cold MAb in preliminary experiments. Viral load measurement Plasma HIV-1 RNA was measured with a Roche Amplicor quantitative RT-PCR kit (Roche, Branchburg, NJ), following the manufacturer instructions. Statistical analysis The Mann Whitney test was used for cross-sectional comparisons of the SBBC with the other cohorts and was perform ed using StatView, version 4.5 for the Macintosh (Abacus Concepts, Berkeley, CA). in members of the SBBC was not significantly different from those of the seronegative control group. However, the proportion of primed (CD45RO 1 ) CD4 1 T lymphocytes was significantly elevated in the SBBC compared with the seronegative control cohort. The absolute number of primed CD4 1 T lymphocytes in the SBBC was slightly higher. The percentage and number of CD4 1 T lymphocytes that were CD38 2 HLA-DR 2 was not significantly different between the SBBC and seronegative cohort. The proportion of CD26 1 CD4 1 T lymphocytes was slightly decreased in the SBBC compared with the CC cohort, although there were no significant differences in the absolute number of this subset. The number of CD8 1 T lymphocytes was significantly elevated in the SBBC compared with the seronegative control group (Fig. 2B). The number of naive (CD45RA 1 CD62L 1 ) CD8 1 T lymphocytes was slightly elevated in the SBBC, compared with the CC control group. The proportion of primed (CD45RO 1 ) CD8 1 T lymphocytes was significantly elevated in the SBBC compared with the seronegative control group, while the number of primed CD8 1 T lymphocytes was significantly elevated. The number of activated CD38 1 HLA-DR 1 CD8 1 T lymphocytes was elevated in the SBBC compared with the seronegative control cohort (Fig. 2B), particularly in those SBBC members with detectable HIV-1 plasma RNA (see below). Similarly, an increase in the number of CD28 2 CD8 1 T lymphocytes in the SBBC compared with the seronegative control group was also observed. RESULTS Comparison of SBBC with HIV-1-uninfected control group CD4 1 T lymphocyte counts in members of the SBBC were comparable to those of the seronegative control group (Fig. 2A). Detailed analysis of subsets showed that the proportion and number of naive (CD45RA 1 CD62L 1 ) CD4 1 T lymphocytes Comparison of subgroups of SBBC members with undetectable versus detectable HIV-1 plasma RNA Three of the six subjects in the SBBC had, 200 plasma HIV- 1 RNA copies per milliliter at all visits (and, 20 copies/ml when tested on at least one visit), while the other three subjects had viral loads ranging from 200 to 4000 copies/ml (Table 1). Neither CD4 1 cell counts, nor the proportion or number of CD45RO 1 CD4 1 T lymphocytes, correlated with viral load within the group (Fig. 2A). However, CD8 1 cell counts for the

4 1522 three subjects with detectable plasma HIV-1 RNA tended to be higher than for the subgroup of SBBC patients with undetectable plasma HIV-1 RNA (Fig. 2B). In particular, the subgroup with detectable viral load had decreased proportions of resting CD38 2 HLA-DR 2 CD4 1 (Fig. 2A) and naive CD8 1 T lymphocytes and elevated numbers of CD45RO 1, CD38 1 HLA-DR 1, and CD28 2 CD8 1 T lymphocytes compared with the subgroup with undetectable HIV-1 RNA (Fig. 2B; p, 0.05 for all comparisons). ZAUNDERS ET AL. Comparisons between SBBC and other LTNPs No significant differences in CD4 1 cell counts or in the number of naive (CD45RA 1 CD62L 1 ) CD4 1 T lymphocytes were observed between the SBBC and TA-LTNP or SA-LTNP cohorts (Fig. 2A). In contrast, an increase in the proportion of primed (CD45RO 1 ) CD4 1 T lymphocytes was found in the SBBC, compared with both TA-LTNPs and SA-LTNPs, with a corresponding increase in the number of primed CD4 1 T lym- A B FIG. 2. Cross-sectional comparisons of circulating CD4 1 (A) and CD8 1 (B) T lymphocytes from SBBC, HIV-1 seronegative, and LTNP cohorts. Subsets are shown as percentages of CD4 1 or CD8 1 T lymphocytes, respectively, and as the corresponding absolute numbers. Results for individual members of the SBBC are shown as either open circles (subjects with undetectable plasma HIV-1 RNA) or closed circles (subjects with detectable plasm a HIV-1 RNA). Box plots depicting median and 10th, 25th, 75th, and 90th percentile values are shown for the HIV-1-seroneg ative, transfusion-acquire d long-term nonprogressor (TA-LTNP) and sexually acquired long-term nonprogressor (SA-LTNP) control cohorts. Significant differences by Mann Whitney test between the SBBC and control cohorts are depicted as * p, 0.05, ** p, 0.01, and *** p,

5 CD4 1 AND CD8 1 T CELLS IN ATTENUATED HIV-1 INFECTION 1523 phocytes. The proportion of CD26 1 CD4 1 T lymphocytes was decreased in the SA-LTNPs compared with the SBBC, although there was no significant difference in the absolute cell counts. The SBBC had comparable numbers of CD8 1 T lymphocytes compared with the TA-LTNP and SA-LTNP cohorts (Fig. 2B). There were no differences in the proportion or number of naive (CD45RA 1 CD62L 1 ) CD8 1 T lymphocytes observed between the SBBC and TA-LTNP or SA-LTNP cohort. The proportion of primed (CD45RO 1 ) CD8 1 T lymphocytes was slightly decreased in the SA-LTNP cohort compared with the SBBC. No differences were observed in the proportion or number of activated CD38 1 HLA-DR 1 CD8 1 T lymphocytes or CD28 2 CD8 1 T lymphocytes between the SBBC and the other LTNP cohorts. 1 1 Longitudinal study of CD45RO CD4 T lymphocytes in the SBBC Five individuals from the SBBC were studied on 6 to 11 occasions over a 3-year period. The results in Fig. 3 show that changes in CD4 1 cell counts were disproportionately due to fluctuations in the number of CD45RO 1 cells, while the number of CD45RA 1 cells appears to be relatively stable over a number of visits. HIV-1-uninfect ed controls were also studied longitudinally over the same time period and Fig. 3 shows that changes in their CD4 1 cell counts were generally due to parallel changes in both subsets. For the five individual SBBC subjects the average fluctuation in CD45RA 1 CD4 1 T lymphocytes was 44 cells/m l per visit (range, 22 to 81 cells/m l), which was twofold lower than for five HIV-uninected controls (mean, 90 cells/m l; range, cells/m l; p, 0.05). Turnover of CD4 1 T lymphocytes was examined directly by intracellular expression of the proliferation marker Ki-67 in CD45RA 1 and CD45RO 1 subsets in three SBBC subjects. An average of 1.8% of CD45RA 1 CD4 1 T lymphocytes (range, %) were Ki-67 1, while in the CD45RO 1 CD4 1 T lymphocyte subset, an average of 4.2% (range, 3.5 5%) were Ki There were no significant differences when compared with four HIV-1-uninfec ted controls (data not shown). Cell surface markers on CD45RO 1 CD4 1 T lymphocytes from SBBC members 1 1 A detailed study of the expression of other markers on the surface of circulating CD45RO CD4 T lymphocytes was undertaken (Fig. 4). In five subjects from the SBBC, CD45RO CD4 T lymphocytes highly coexpressed CD29 (present on 69 92% of CD45RO CD4 T lymphocytes) and partly coexpressed CD11a bright (30 78% of CD45RO CD4 T lymphocytes) and CD62L (37 55%). A minority of CD45RO CD4 T lymphocytes coexpressed CD25 (6 19% of CD45RO CD4 T lymphocytes), HLA-DR (6 20%), and CD38 (10 23%). Sim- FIG. 3. Longitudinal analysis of CD4 1 T lymphocytes: representative total CD4 1 T lymphocyte (m ), CD45RO 1 CD4 1 T lymphocyte (d ), and CD45RA 1 CD4 1 T lymphocyte (s ) cell numbers for three SBBC subjects and three HIV-negative controls. The x axes represent the time span of this study (May 1995 July 1998). The y axes represent the cell counts for each subset (cells/ m l).

6 1524 ZAUNDERS ET AL FIG. 4. Flow cytometric analysis of additional cell surface markers on CD3 CD4 CD45RO lymphocytes: representative histograms for subject C135 showing percentages of CD3 CD4 lymphocytes that are CD45RO and positive or negative for CD29, CD11a, CD62L, CD25, HLA-DR, and CD38. ilar results for these markers were obtained for CD45RO 1 CD4 1 T lymphocytes from four HIV-1-uninfec ted controls (data not shown). DISCUSSION The main objective of these investigations was to study the composition and activation of CD4 1 and CD8 1 T lymphocytes after long-term infection with a strain of HIV-1 with a natural deletion in the nef/ltr region of the genom e. Importantly, our results show that the SBBC members had numbers of naive CD4 1 and CD8 1 T lymphocytes comparable to the levels in matched HIV-1-negativ e control groups. A decrease in this subset of circulating T lymphocytes is associated with progressive HIV-1 infection 9 and is generally refractory to antiretroviral therapy. 12,14 However, SBBC recipients had an elevated proportion of primed (CD45RO 1 ) CD4 1 T lymphocytes by comparison with age-, sex-, and transfusion-mat ched HIV-1-seroneg ative controls, and substantial elevations in proportions and numbers of CD45RO 1 CD4 1 T lymphocytes compared with two other LTNP cohorts. The proportion of CD45RO 1 CD4 1 T lymphocytes within the SBBC was not correlated with age, and the increased proportion was seen in comparisons with agematched controls, suggesting this was not simply due to age. 28 Also, the increase in the proportion of CD45RO 1 CD4 1 T lymphocytes was not due to a decrease in the number of CD45RA 1 CD4 1 T lymphocytes, as occurs in late-stage disease. 9,10 The CD45 glycoprotein is a transm embrane protein tyrosine phosphatase that is required for T cell activation via the T cell receptor. 29 The low molecular weight splice variant CD45RO of CD45 is expressed on activation, 30 but reversion of at least a proportion of these cells to a CD45RA 1 phenotype can occur in vivo over a period of weeks to months, 31 dependent on no further exposure to antigen. 32 The detailed phenotype of the CD45RO 1 CD4 1 T cells from the SBBC, showing expression of CD29 and CD11a brigh t (LFA-1 bright ) and partial loss of CD62L, is consistent with the interpretation that these cells have recently responded to antigen. 32,33 We have already shown that peripheral blood mononuclear cells (PBMCs) from the SBBC exhibited normal in vitro proliferative responses to recall antigens, as well as responses to HIV-1 antigens. 8 The results presented here are consistent with these in vitro studies, since lymphoprolifera tive responses to recall antigens have been attributed to CD45RO 1 CD4 1 T lymphocytes. 34 Fluctuations in CD4 1 cell counts over time for the SBBC were generally due to changes in CD45RO 1 cell numbers, exhibiting patterns of subset changes that appeared to differ from those of HIV-1-uninfec ted controls. We have described shortterm increases in CD4 1 CD45RO 1 cells in patients treated with protease inhibitor, 12 believed to represent the rapid appearance of preexisting entrapped cells, 12,13 suggesting that trafficking of CD4 1 CD45RO 1 cells contributes greatly to changes in their

7 CD4 1 AND CD8 1 T CELLS IN ATTENUATED HIV-1 INFECTION concentration in peripheral blood. Consistent with this hypothesis, the turnover of CD4 CD45RO cells, as measured by expression of the nuclear antigen Ki-67, a marker of recent proliferation, 35 was not significantly different in the SBBC compared with controls. Conversely, fluctuations of CD45RA CD4 T lymphocytes were reduced in the SBBC and further study is required to determ ine whether this is restricted to the SBBC or is a common result of HIV-1 infection, possibly reflecting an effect on normal thymic output. 36,37 It is also possible that CD45RO CD4 T lymphocytes are continually being lost in most HIV-1-infected patients. This subset is more sensitive in vitro to the cytopathic effects of HIV and several studies have reported a decrease in the proportion of CD4 T lymphocytes that express CD45RO in asymptomatic HIV-1-infecte d subjects. 10,43,44 Replication of HIV-1 in CD45RO CD4 lymphocytes in vitro was shown to be decreased by specific deletion of the nef gene, 41 and loss of CD4 cells in adult macaques infected with SIV has been linked to expression of Nef protein. 45 Therefore, it is possible that in the particular case of the SBBC, a reduced cytopathic effect of the attenuated HIV-1 in vivo combined with chronic immune activation has led to an increased proportion of CD45RO CD4 T lymphocytes. A third, but not mutually exclusive, possibility is that CD45RO CD4 T lymphocytes in the SBBC include actively maintained, HIV-1-specifi c cells. Results in a model of chronic herpesvirus infection in mice showed that continued CD8 T cell control of infection was dependent on the presence of CD4 T cells. 46 The importance of CD4 cell function in the control of HIV-1 infection has been suggested by the low HIV-1 burden in a small number of LTNPs with vigorous in vitro HIVspecific CD4 cell proliferative responses. 47 Another study has described a small group of LTNP patients with particularly low viral load and high CD4 cell counts without prominent CD8 HIV-1-specific responses. 25 In this regard, studies of precursor frequencies for HIV-specific CD4 T cells in the SBBC may be informative. The proportion of CD26 CD4 T lymphocytes was slightly, but not significantly, decreased in the SBBC compared with age-, sex-, and transfusion-mat ched HIV-1-seroneg ative controls. However, the degree of reduction of this subset was found to be much greater in the sexually acquired LTNP cohort, in line with a previous study, 15 and further suggests that the SBBC differs from other LTNP cohorts. The presence of CD38 HLA-DR and CD28 CD8 T lymphocytes appeared to be a sensitive marker of viral burden within the group of SBBC patients, consistent with reports of the correlation of these subsets with plasm a HIV-1 RNA concentrations. 48,49 Furthermore, we have now shown that three patients with minimal viral burden had levels of activated CD8 T lymphocytes that were within the normal range, similar to patients who have recovered from acute infection with Epstein Barr virus (EBV). 50 This observation is of particular relevance to those patients treated with highly active antiretroviral therapy and who clear plasma viremia but who have not completely normalized CD8 cell activation, 49,51 suggesting that residual activation is indicative of low level viral replication. Other studies have shown that highly purified CD38 HLA- DR 18 and CD28 19,20 CD8 lymphocytes contained HIV-specific CTL activity. Studies of HIV-1-specific CTLs in SBBC members have shown that three recipients had strong CTL activity (C49, C18, and C98), while two had weak activity (C54 and C64) and CTLs were absent in one (C135). 6 It is surprising, therefore, that one patient with undetectable viral load and normal levels of CD8 1 cell activation (C49) had strong CTL activity, while one patient with detectable viral load and elevated levels of CD8 1 cell activation had weak CTL activity (C54). These results suggest that activation of CD8 1 T lymphocytes in vivo is most closely related to viral burden and does not necessarily reflect in vitro CTL activity in this group of patients. Interestingly, also, the three individuals with normal levels of CD8 1 cell activation had stable CD4 1 cell counts, while two of the three individuals with elevated activated CD8 1 T lymphocytes had slow, but significant, decreases in CD4 1 cell counts. 3 In conclusion, long-term infection of the SBBC with an attenuated quasi-species of HIV-1 has not led to significant loss of naive CD4 1 or CD8 1 T lymphocytes, in common with other LTNPs included in this study. However, members of the SBBC have an increase of primed CD4 1 T lymphocytes that distinguishes them as a group from other HIV-infected subjects, including other LTNPs. These results suggest the possibility of a decreased in vivo cytopathic effect of nef/ltr mutant HIV-1 on primed CD4 1 T lymphocytes, similar to that described in vitro, and in adult macaques infected with attenuated SIV. However, in the light of findings of disease progression resulting from long-term infection with attenuated SIV 7 and HIV-1, 52 our report 3 suggests that the SBBC viral strain can in fact cause immunological decline in some individuals. While the recipients with detectable plasma viral RNA described in this study continue to remain asymptomatic, the donor member of the SBBC has progressed to AIDS and has commenced antiretroviral therapy. 3 We cannot rule out that a similar immunologica l decline may occur in the SBBC recipients, and we suggest that this nef/ltr-defective HIV-1 strain will not form the basis of a live attenuated vaccine. ACKNOWLEDGMENTS This work was supported in part by an HIV R&D Syndicate and a Commonwealth AIDS Research Grant administered by the National Health and Medical Research Council of Australia. We thank Leon McNally, Sara Hancock, Claire Temby, and Virginia Nink for help with preparation and analysis of samples. 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