during the course of hiv-1 infection , multifactorial t - lymphocyte ( t - cell)-mediated mechanisms contribute to the progressive loss of host immune function [ 15 ] . in infected individuals ,
immune dysregulation occurs early and is characterized by a decrease in cd4 + cell count , a concurrent rise in cd8 + cells , a progressive decline in the cd4+/cd8 + ratio , and defective thymocyte proliferation . during late - stage disease , loss of t - cell homeostasis also occurs [ 7 , 8 ] .
t cells are chronically activated throughout the course of hiv infection , as indicated by an increase in the expression of the antigens ki67 ,
cd38 , and human leukocyte antigen ( hla)-dr , with cd38 recognized as the most reliable marker of immune activation [ 13 , 5 , 9 ] .
immune activation provides the virus with a steady pool of target cells and has been linked with increased polyclonal t - cell proliferation and turnover , as well as increases in the apoptotic marker cd95 [ 1013 ] and activation - induced cell death [ 12 , 1416 ] .
concomitant with the decline of cd4 cells in the peripheral blood , the frequency of the cd4 + cd28 null subset increases with disease progression and eventual progression to aids .
the presence of cd28 on t cells is critically important for the generation of t - cell responses .
interaction of this costimulatory molecule with its ligands increases the expression of antiapoptotic proteins and improves interleukin ( il)-2 production .
the increase in circulation of t cells with a cd4 + cd28 null phenotype is consistent with a process known as replicative senescence [ 11 , 13 , 1719 ] .
t cells that lack cd28 surface expression are nonanergic , oligoclonally expanded , and terminally differentiated , with limited replicative capacity and increased sensitivity to apoptosis [ 20 , 21 ] .
these alterations in phenotype are accompanied by cytokine changes consistent with a chronic proinflammatory state .
the failure to produce adequate amounts of il-2 leads to a marked functional impairment of cellular and humoral immunity .
reduced il-2 expression has been associated with a shift from th1 cytokine responses to th2 cytokine responses .
the dysregulation of cytokine secretion during the course of hiv infection has been examined using a wide range of methods , including enzyme - linked immunosorbent assay , reverse transcriptase - polymerase chain reaction , t - cell cloning , and , more recently , flow cytometric intracellular cytokine detection
. of these methods , only flow cytometry allows quantitative and qualitative determination of cytokine expression patterns in individual t cells .
successful antiretroviral ( arv ) therapy results in improvements in circulating cd4 + t - cell levels with decreases in cd8 + counts and declines in immune activation and cd95 expression [ 15 , 7 , 9 , 2226 ] .
limited data on replicative senescence suggest a lack of significant improvement in the short term , despite adequate hiv suppression .
some studies have suggested that protease inhibitor- ( pi- ) based regimens may have a greater effect on cd4 + count recovery than nonnucleoside reverse transcriptase inhibitor - based regimens [ 27 , 28 ] .
some of the previous studies on immune recovery with arv therapy have been cross - sectional in design , conducted primarily in white males , and focused on absolute cd4 + count or cd4+% recovery .
furthermore , most of these studies did not comprehensively assess the functional aspects of immune recovery .
of the studies that did evaluate the influence of arv therapy on functional immune parameters , most were drug class specific [ 3032 ] .
grace ( gender , race , and clinical experience ) is the largest arv trial to focus on women with hiv-1 in north america and was designed to assess sex - based and race - based differences in efficacy , safety , and tolerability of the pi darunavir with low - dose ritonavir ( drv / r ) plus an optimized background regimen over 48 weeks in a diverse , treatment - experienced patient population .
the aim of this prospective substudy was to quantitatively and qualitatively evaluate the recovery of functional immunity ( t - cell function ) with a drv / r - based regimen in a subset of patients from the grace study .
grace was a multicenter , open - label , single - arm , phase iiib study conducted at 65 sites across usa , puerto rico , and canada that enrolled 429 treatment - experienced patients ( viral load  1000 hiv-1 rna copies / ml ) aged at least 18 years with documented hiv-1 infection .
patients received drv / r 600/100  mg twice daily plus an investigator - selected background regimen that could include etravirine .
patients at participating grace study sites were eligible for the prospective immunology substudy , which aimed to enroll up to 100 subjects .
all subjects were required to sign a separate , independent ethics committee / institutional review board ( irb ) informed consent form specific to this substudy .
the irbs were quorom review inc . , seattle , wa , office of human research ethics 
unc irb , chapel hill , nc , and aids research consortium of atlanta , atlanta , ga . each irb approved of this study .
the study was conducted in accordance with the principles of the declaration of helsinki and followed good clinical practice guidelines .
viral suppression in this analysis was defined as achieving hiv-1 rna less than 50 copies / ml at week 48 .
immune function and phenotype were determined by flow cytometry at baseline and weeks 12 and 48 in virologically suppressed and nonsuppressed patients .
changes in immune phenotype were determined from subsets of cd4 + and cd8 + t cells , with immune activation defined as increased expression of t - cell cd38 and hla - dr surface markers , and immune replicative senescence by increased frequency of circulating cd28 null t cells .
changes in immune function were assessed by lymphocyte proliferation in response to candida and tetanus ( recall antigens ) , phytohemagglutinin ( pha ) and pokeweed ( mitogenic plant lectins ) , and cd3+/cd28 + and by intracellular cytokine expression of il-2 , interferon - gamma ( ifn- ) , and tumor necrosis factor - alpha ( tnf- ) in response to staphylococcal enterotoxin b. a whole blood lysing technique was used and subpopulations were assessed according to a standard protocol for 3-color and 4-color immunofluorescence flow cytometry using fluorochrome conjugated monoclonal antibodies and a fluorescence activated cell sorter .
a panel of monoclonal antibodies was used to delineate total cd3 + , total cd4 + and cd8 + t cells , cd4+/cd8 + ratio , activated t cells ( cd38+/hla - dr+ ) , immune replicative senescence ( cd4+/cd28 + or cd8+/cd28 + ) , and apoptotic cells ( cd95 + ) . the vybrant cfda se cell tracer kit ( molecular probes , inc . )
lymphocytes were labeled with carboxyfluorescein diacetate and succinimidyl ester cfda se and then incubated at 37c with 5% carbon dioxide for 7 days .
the label is inherited by daughter cells after division . labeled lymphocytes with carboxyfluorescein diacetate and succinimidyl ester were detected by flow cytometry using cd45 markers for gating strategy .
the reagents used were candida , tetanus , cd3+/cd28 + , pha , and pokeweed .
flow cytometric intracellular cytokine detection of th1 cytokines including ifn- , il-2 , and tnf- was assessed using the bd cytofix / cytoperm plus fixation / permeabilization ( golgiplug protein transport inhibitor ; bd biosciences ) .
peripheral blood mononuclear cells were stimulated with staphylococcal enterotoxin b ( sigma ) and a cef control peptide pool ( cmv , ebv , influenza virus ; nih reagent 9808 ) .
then , surface and intracellular staining antibodies were added in a single staining step ( anti - hu - ifn-/cd69/cd4/cd3 , anti - hu - il2/cd69/cd4/cd3 , anti - hu - tnf-/cd69/cd4/cd3 ; bd biosciences ) .
a total of 32 patients with hiv-1 were enrolled in the substudy ; 25 ( 78% ) patients had week 48 data and 19 ( 59% ) were virologically suppressed at week 48 .
the normal comparator group consisted of 34 healthy , hiv - seronegative individuals , 17 ( 50% ) of whom were women and 25 ( 74% ) of whom were white .
patient demographics and baseline characteristics for total and virologically suppressed patients are shown in table 1 . the median ( range )
cd4 + count at baseline for the total patient population was 191 ( 2 , 463 ) and the median ( range ) cd4 + count in virologically suppressed patients at baseline was 222 ( 2 , 398 ) cells / mm ( table 1 ) . at week 48 , the median ( range ) cd4 + count for the total population and virologically suppressed patients was 337 ( 98 , 812 ) and 398 ( 119 , 812 ) cells / mm , respectively .
in virologically suppressed patients , the cd4+% increased significantly from baseline to week 48 ( p < .03 ; figure 1 ) .
the median ( range ) cd8 + count at baseline for the total patient population was 912.5 ( 288 , 3131 ) cells / mm , while the median ( range ) cd8 + count in virologically suppressed patients at baseline was 1037 ( 288 , 3131 ) cells / mm .
the cd8+% decreased significantly from baseline to week 48 ( p < .01 ) in virologically suppressed patients ( figure 1 ) .
the median ( range ) cd4+/cd8 + ratio at baseline was 0.22 ( 0.01 , 0.70 ) in all patients and 0.22 ( 0.01 , 0.70 ) in virologically suppressed patients .
the cd4+/cd8 + ratio at weeks 12 and 48 is displayed in figure 1 and table 2 .
the cd4+/cd8 + ratio increased significantly from baseline to week 48 ( p < .01 ) in suppressed patients .
the percentage of cd4 + and cd8 + cells at weeks 12 and 48 in suppressed patients is shown over 48 weeks in figure 1 .
the percentage of cd4 + cells significantly increased ( p < .01 ) and cd8 + cells significantly decreased ( p = .03 ) from baseline to week 48 .
improvements in immune activation , as measured by decreases in cd38 and hla - dr expression on cd4 + and cd8 + cells over the course of the study , were observed in both the total patient population ( table 2 ) and in virologically suppressed patients ( table 2 ; figure 2 ) .
the percentage of apoptotic ( cd95 + ) cd4 + cells in suppressed patients significantly increased from baseline to week 48 ( p = .0142 ; table 2 ; figure 2 ) .
the percentage of apoptotic cd8 + cells , on the other hand , significantly decreased from baseline to week 48 in suppressed patients ( p = .0025 ; table 2 ; figure 2 ) .
changes in immune replicative senescence were measured by changes in the frequency of cd4+/cd28 or cd8+/cd28 cells ( table 2 ) .
there was little change in the expression of costimulatory marker cd28 + on cd4 + cells from baseline to week 48 in the total patient population or the virologically suppressed group ( table 2 ) .
there was a small decrease in the expression of cd28 + on cd8 + cells in the total patient population and the virologically suppressed population from baseline to week 48 ( table 2 ) .
the ability of cd4 + lymphocytes to respond to mitogens and recall antigens improved in grace patients over the course of the study .
proliferation in response to cd3+/cd28 + and pha was at , or near , normal levels by week 12 in virologically suppressed patients , and proliferation in response to pokeweed and candida was at normal levels by week 48 ( figure 3 ) .
tumor necrosis factor - alpha and il-2 significantly increased in staphylococcal enterotoxin b - stimulated cd4 + cells of virologically suppressed patients by week 48 ; there was no significant change in ifn- in the stimulated cd4 + cells ( figure 4 ) .
few published studies within clinical trials have prospectively assessed in vitro changes in immune function as measured by lymphocyte proliferation [ 34 , 35 ] , and none of these assessed intracellular cytokine production in response to arv therapy .
this substudy from the grace trial evaluated t - cell function in a racially diverse , treatment - experienced population comprised of more than 30% women .
as expected , based on results from previous studies [ 24 , 27 , 28 ] , drv / r - based therapy resulted in increases in cd4 + cell counts and decreases in cd8 + counts in virologically suppressed patients , with an improved cd4+/cd8 + ratio .
in addition , we found that drv / r - based arv therapy was associated with progressive functional immune recovery over 48 weeks in virologically suppressed patients , as demonstrated by improved lymphocyte response to mitogens and recall antigens .
this suggests that not only do the cd4 + cell counts of hiv-1-infected patients improve during drv / r - based therapy , but their ability to respond in vitro to immune stimuli may improve as well .
results from this report are consistent with a recent study that demonstrated significant decreases in immune activation and apoptosis in cd4 + and cd8 + t cells and a decrease in immune cd8 + senescence following arv therapy .
while there have been studies evaluating the influence of arv therapy on immune parameters , data typically represent class - specific rather than regimen - specific treatment approaches .
for example , therapy with integrase inhibitors has been shown to result in larger cd4 + cell count increases compared with other arv classes [ 29 , 37 , 38 ] .
the work shown here represents a regimen - specific study , with combination drv / r treatment in addition to an optimized background regimen that could differ across patients .
unfortunately , we can not assess differences between regimens , as our study was not comparative .
the reduction in t - cell activation seen here has previously been shown to correlate with the reduction in viral load , as well as an improved response to recall antigens .
the observed hiv-1-induced increase in apoptotic cd95+/cd4 + cells has been suggested to be independent of arv activity ; the pi saquinavir has been shown to decrease cd95 expression in cd4 + cells from healthy donors whose cells were previously briefly incubated with hiv-1 virus .
although anti - cd95-induced apoptosis declines with therapy , this change is only occasionally associated with a reduction in expression of cd95 on t lymphocytes .
it is thus not surprising that in our study cd95 expression on t cells did not decline . in the grace study ,
no improvement in the cd4+/cd28 or cd8+/cd28 phenotype was seen , consistent with other studies that have suggested that cd28 expression is not normalized in hiv-1infected patients who have undergone up to 3 years of arv therapy [ 40 , 41 ] .
one possible limitation to this study is the small sample size , which may limit data interpretation .
the study aimed to enroll 50 women and 50 men , but , due to a delayed start date , after initiation of the grace study , the patient pool for this substudy was limited and the enrollment goal was not reached .
nonetheless , the data obtained are similar to those of other trials . both immune phenotype and function of cd4 + and cd8 + cells
were significantly improved in treatment - experienced patients receiving drv / r - based therapy , as evidenced by positive changes in the capacity to proliferate and the expression of intracellular cytokines by cd4 + cells .
the functional recovery observed in virologically suppressed patients , as assessed by proliferative response and intracellular cytokine expression , was also seen in nonsuppressed patients , although to a lesser degree .
antiretroviral therapy has been available for 25 years ; however , there has been limited research on immune recovery after long - term treatment .
furthermore , the focus of immune recovery has historically been an assessment of cd4 + cell counts . in the current study , in addition to the evaluation of cd4 + cell counts ,
cd4 + cell activation ( cd38+/hla - dr+ , cd95 + ) , function , and senescence ( cd28 ) were measured as well as cd8 + cell counts , activation , function , and senescence .
it should be noted that , as observed in this trial , despite the improvements in immune phenotype and function seen with arv therapy , complete normalization of cd4 + and cd8 + parameters is rarely achieved in hiv-1-infected individuals [ 24 , 42 ] .
however , we hypothesize that there may be some clinical benefit even in patients who do not experience measurable or significant increases in cd4 + cell counts .
it is feasible that , in patients with higher baseline cd4 levels , measurable increases in cd4 + cell counts may not be observed .
previous studies have shown that despite cd4 + cell count normalization , the pretreatment nadir cd4 + count and level of cd28 cd4 + coexpressing lymphocytes determine ongoing immune competence , including responses to immunization . despite the successful hiv therapy noted in the grace substudy
this possible treatment - resistant expansion and persistence of cells with a senescence phenotype may have potential for long - term cardiovascular , metabolic , and other aging - associated consequences .
this grace substudy demonstrates that drv / r - based therapy improved cd4 + cell recovery and was associated with progressive functional immune recovery over the 48-week study period .
thus , initiation of arv therapy may be required not only to restore immune function , but also to diminish the effects of chronic inflammation .
 
 objective . during the course of hiv infection , 
 progressive immune deficiency occurs . 
 the aim of this prospective substudy was to evaluate the recovery of functional immunity in a subset of patients from the grace ( gender , race , and clinical experience ) study treated with a drv / r - based regimen . 
 methods . the recovery of functional immunity with a darunavir / ritonavir - based regimen was assessed in a subset of treatment - experienced , hiv-1 infected patients from the grace study . results . 
 19/32 patients ( 59% ) enrolled in the substudy were virologically suppressed ( < 50 copies / ml ) . in these patients , 
 median ( range ) cd4 + cell count increased from 222 ( 2 , 398 ) cells / mm3 at baseline to 398 ( 119 , 812 ) cells / mm3 at week 48 . 
 cd8+% decreased significantly from baseline to week 48 ( p = .03 ) . 
 proliferation of cd4 + lymphocytes in response to cd3+/cd28 + , phytohemagglutinin , and pokeweed was significantly increased ( p < .01 ) by week 12 . 
 proliferation in response to candida and tetanus was significantly increased by week 48 ( p < .01 and p = .014 , resp . ) . 
 staphylococcal enterotoxin b - stimulated tumor necrosis factor - alpha and interleukin-2 in cd4 + cells was significantly increased by week 12 ( p = .046 ) and week 48 ( p < .01 ) , respectively . conclusions . 
 darunavir / ritonavir - based therapy demonstrated improvements in cd4 + cell recovery and association with progressive functional immune recovery over 48 weeks . 
 this trial is registered with nct00381303 . 