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Limitations of current antiretroviral therapy in HIV-1 infection: the search for new
strategies
Sankatsing, S.U.C.
Publication date
2004
Link to publication
Citation for published version (APA):
Sankatsing, S. U. C. (2004). Limitations of current antiretroviral therapy in HIV-1 infection: the
search for new strategies.
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Noo detrimental immunological effects of
mycophenolatee mofetil and HAART in treatment
naïvee acute and chronic HIV-1 infected patients
N.Vrisekoop,, S.U.C. Sankatsing, C A Jansen, M.T.L. Roos,
S.. A. Otto, H. Schuitemaker, J.MA Lange, J.M. Prins,
F.. Miedema
Abstract t
Background:Background: Mycophenolate mofetil (MMF) has been proposed for HIV-1 therapyy because of its guanine depleting effect, which is expected to interfere
withh HIV-1 replication directly by hampering reverse transcription and indirectlyy via inhibition of CD4+ T cell proliferation. However, MMF treatment mightt also compromise lymphocyte reconstitution and HIV-specific immunity.
Objective:Objective: To study the effects of MMF in combination with HAART on T cell proliferation,, lymphocyte reconstitution and HIV-specific CD4+ and CD8+ T cell
responses. .
Methods:Methods: We longitudinally studied the immunological effects of MMF treatmentt in combination with HAART in 6 therapy naive, acute or chronic
HIV-11 infected patients, as compared to 8 patients treated with HAART alone.
Results:Results: T cell proliferation in whole blood cultures (WBC) of patients treated withh MMF was inhibited. Strikingly, ex vivo Ki67 expression within T cells was nott influenced by MMF treatment. In vitro studies showed that Ki67 expressionn was induced at an early step of the cell cycle not inhibited by guaninee depletion. When MMF treatment was stopped a transient increase in apoptosiss and Ki67 expressing T cells was observed. Treatment with MMF hadd no effect on the increase in absolute numbers of CD4+ T cells, or on HIV-specificc T cell responses.
Conclusion:Conclusion: Increased Ki67 expression after discontinuation of MMF together withh near complete inhibition of WBC during MMF treatment indicated that T
celll proliferation was inhibited in patients treated with MMF. Still, there was no evidencee for detrimental effects of treatment with MMF in addition to HAART onn lymphocyte reconstitution or HIV-specific immunity.
Introduction n
Itt has proven impossible to eradicate HIV-1 with currently available antiretrovirall therapies. Beside a latent viral reservoir that is maintained in non-replicatingg cells 1_4, a low level of viral replication is sustained even in individualss in whom highly active antiretroviral therapy (HAART) has decreasedd the plasma viremia to undetectable levels 5"10. A logical approach too improve treatment of HIV-1 is to directly or indirectly interfere with the availabilityy of target cells and target cell characteristics necessary for completionn of virus replication. Although T cell proliferation, assessed by the expressionn of the proliferation marker Ki67, decreases by HAART, it remains elevatedd compared to normal values throughout 48 weeks of treatment11. To reducee residual replication and reseeding of the latent reservoir6 even further, lymphocytee proliferation is thus a likely target for treatment.
Basedd on this idea, HIV-1 infected patients have been treated with the immunosuppressantt hydroxyurea (HU), an inhibitor of DNA synthesis. Indeed itt has been shown that treatment with HU inhibits HIV-1 replication in lymphocytess in vitro and acts synergistically with the nucleoside analogue didanosinee (ddl)12. Furthermore, adding HU to treatment with ddl decreased thee plasma virus load in vivo13"15, arguing for a role of target cell availability in limitingg HIV-1 infection 16. However, along with other toxicities, additional treatmentt with HU blunts the increase or even causes a decrease in the absolutee number of CD4+ T cells 14,15'17.
Thee immunosuppressant mycophenolate mofetil (MMF) is a prodrug of mycophenolicc acid (MPA), its active metabolite, which selectively suppresses lymphocytee proliferation. MPA suppresses lymphocyte proliferation through non-competitive,, reversible inhibition of inosine 5'-monophosphate dehydrogenasee (IMPDH), a key enzyme in the de novo synthetic pathway of thee purine guanine 18-19. inhibiting IMPDH results in a decrease of available guaninee with the result that lymphocytes fail to progress to the S phase of the celll cycle 20"22. Whereas T and B lymphocytes strongly depend on de novo synthesiss of purines for their proliferation, other cell types are able to make usee of both de novo synthesis and the salvage pathway. Therefore, MPA is
believedd to have a more potent cytostatic effect on lymphocytes than on other celll types.
MMFF has been proposed for HIV-1 therapy, since guanine depletion is expectedd to prevent HIV-1 replication indirectly via inhibition of CD4+ T cell divisionn and because MPA suppressed HIV-1 replication directly by hamperingg reverse transcription in vitro 23. Furthermore, the efficacy of the guanosinee analogue reverse transcriptase inhibitor abacavir increases in the presencee of MPA 24. Indeed Chapuis et al. demonstrated that the addition of MMFF to HAART in HIV-1 patients with a plasma HIV-1 RNA of less than 5 copies/mll for over 36 weeks resulted in a decrease of the number of latently infectedd CD4+ T cells. Moreover, they reported that MMF treatment resulted in aa minor decrease in the percentage of dividing CD4+ and CD8+ T cells as measuredd by Ki67 expression 25. On the other hand, inhibiting lymphocyte proliferationn by MPA might also inhibit the immune response against HIV-1, causingg decreased clearance of the virus. In addition, lymphocyte counts couldd be negatively influenced by MPA, with the risk of reducing or preventing lymphocytee reconstitution.
Wee studied the effects of combined treatment with MMF and a triple class, five-drugg regimen on T cell proliferation, lymphocyte reconstitution and HIV-1 specificc T cell responses in therapy naive, chronic and acute HIV-1 infected patientss compared to patients treated with HAART alone.
Materiall and methods
Patients Patients
Fourteenn antiretroviral naïve men, 7 with an acute infection (treatment started withinn 2 months after onset of symptoms of acute HIV infection) and 7 with a chronicc infection (treatment started when CD4+ T cell numbers were <350 cells/ull or when plasma HIV-1 RNA was >30,000 copies/ml) were recruited. Thee Medical Ethics Committee of the Academic Medical Center approved the studyy and written informed consent was obtained from all patients. A detailed descriptionn and baseline characteristics have been reported elsewhere 26. All patientss started with a triple class, five-drug regimen consisting of didanosine, abacavir,, lamivudine, indinavir (boosted with ritonavir) and nevirapine.
Patientss were randomized to one group with MMF and one group without MMF.. Patients 002, 003 and 004 started with 500 mg MMF twice daily and afterr 4 weeks the dose was increased to 1000 mg twice daily. However since thee plasma MPA levels were well above the minimum required concentration
27
,, the dose was decreased to 500 mg twice daily after 12-24 weeks of treatmentt and patients 008, 011 and 014 who were also randomized to MMF usedd 500 mg twice daily. Patients were seen before study entry, at the start of therapy,, and at different time intervals during therapy, for clinical assessments andd for obtaining blood samples. Patient 012, who did not use MMF, stopped aill medication after 19 weeks because of elevated liver enzymes and restartedd all medication except nevirapine after normalization of his liver enzymess at week 25. Patient 011 discontinued MMF at week 16 at his own requestt because of a sore throat. Overall, no more side effects were seen in patientss using MMF.
WholeWhole blood cultures
Thee whole blood lymphocyte culture is described in detail elsewhere 28. Briefly,, ex vivo proliferative responses to aCD3 and aCD28 (Sanquin reagents,, Amsterdam, The Netherlands) in 10 times diluted heparanized venouss blood were measured after 4 days of culture by means of 3 H-thymidinee incorporation, added 24 hr before harvesting. Reactivity was calculatedd back to counts per minute (cpm) per 103 CD3+ T cells and results aree expressed as percentages of the mean response in T cells from 3 healthy controlss in parallel cultures.
FlowFlow Cytometry
CD4++ and CD8+ T cell numbers were measured in peripheral blood using immunofluorescencee flow cytometry. Peripheral blood mononuclear cells (PBMC)) were obtained by Ficoll-Paque density gradient centrifugation from heparinizedd blood. Naive (CD27+CD45RA+), CD27+ memory (CD27+CD45RA" \\ CD27' memory (CD27CD45RA) and effector (CD27CD45RA+) CD4+ and CD8++ T cell numbers were defined as described previously and assessed by floww cytometry 29,3 .
Peripherall blood T cell proliferation in CD4+ and CD8+ T cell subsets was studiedd by flow-cytometric measurements of Ki67 nuclear antigen, as describedd previously 29~31. Briefly, cryopreserved PBMCs were thawed and incubatedd with monoclonal antibodies (mAb) to CD4- or CD8-PerCP, CD45RO-PEE (Becton Dickinson (BD), San Jose, California) and biotinylated CD277 (Sanquin Reagents). After washing, cells were incubated with anti-Streptavidin-APCC (BD). Next, cells were fixated (FACS Lysing Solution, BD) andd permeabilized (FACS Permeabilisation Buffer, BD). Lymphocytes were stainedd intracellular^ with Ki67-FITC (Novocastra laboratories, Newcastle, Unitedd Kingdom), after which cells were fixed using Cellfix (BD) and analyzed onn a FACSCalibur (BD) with Cellquest software. Apoptosis was measured by FACSS analysis using AnnexinV and Propidium Iodide staining according to manufacturer'ss protocol (Bender MedSystems, Vienna, Austria).
HIV-1HIV-1 specific immune responses
Too measure IFNy producing HIV-1 specific T cells, frozen PBMCs were thawedd and cells were aliquoted at 2x106 cells per ml. CD8+ T cells were stimulatedd with the following peptides: HLA-A2 restricted peptides SLYNTVATLL (Gag) and ILKEPVHGV (Pol) and HLA-B8 restricted peptides EIYKRWMM (Gag) and FLKEKGGL (Nef) (Dutch Cancer Institute) in a concentrationn of 1 ug/ml. CD4+ T cells were stimulated with either gag or nef pooledd peptide pools (15 mers with 11 overlap, HXB2, AIDS Reagents) in the presencee of co-stimulation (2ug/ml aCD28 (CLB) and 2 ug/ml aCD49d (BD Pharmingen,, San Diego, California)). The concentration of the individual peptidess within each pool was 2 ug/ml. As a positive control stimulation with PMA77 lonomycin was used. Stimulations were performed for 4 hours (CD8+ T cells)) or 6 hours (CD44 T cells) at . After 1 hour, Brefeldin A (BD) was addedd to block extracellular secretion of cytokines. Next, cells were fixed and permeabilized.. Subsequently CD8+ T cells were stained with mAb to CD8-PerCPP and IFN-^FITC (BD) and CD4+ T cells with mAb to CD3-PerCP, CD4-APCC and IFN-y-FITC (BD). Cells were then fixed and analyzed using a FACSCaliburr flow cytometer (BD).
IFNyy producing HIV-1 specific CD8+ T cells were enumerated using IFNy-specificc ELIspot assays for patients with HLA-A3 and HLA-B7, as previously describedd 3233 using IFNy-specific mAbs (MABTECH, Stockholm, Sweden). PBMCss were added at 105 cells per well in triplicate in the presence of 10 ug/mll peptide, and incubated overnight at . PHA stimulation served as a positivee control. IFNy-producing cells were counted using an automated spot readerr (AEL.VIS., Software version 3.2, Hanover, Germany). Only samples withh at least twice the number of spots observed with media alone were consideredd positive. Peptides used were: HLA-A3 restricted peptides QVPLRPMTYKK (Nef) and RLRPGGKKK (Gag) (LUMC, Leiden, Netherlands) andd HLA-B7 restricted peptides IPRRIRQGL (Env gp41), RPNNNTRKSI (Env gp120)) and TPGPGVRYPL (Nef) (Dutch Cancer Institute, Amsterdam, Netherlands).. Frequencies of IFNy producing cells were reported after subtractionn of the frequencies in medium controls.
InIn vitro thymidine incorporation and Ki67 expression
Afterr thawing PBMC of healthy bloodbank donors, cells were cultured in mediumm supplemented with 10% Fetal Calf Serum. MPA (Gibco BRL, Grand Island,, NY) was added in a concentration of 3 ug/ml and subsequently the cellss were stimulated with aCD3 and aCD28. Unstimulated cells served as a negativee control and cells stimulated with aCD3 and aCD28 only were used ass a positive control. Proliferative responses were measured in triplicate after 0,, 4, 7 and 8 days of culture by means of incorporation of 3H-thymidine (added 16hh before harvesting). At day 0, 4 and 6 Ki67 expression was determined withinn CD4+ and CD8+ lymphocytes by flow-cytometric measurements.
CellCell separation and TREC analysis
CD4++ and CD8+ T cells were purified from thawed PBMC by magnetic bead separation,, using the MiniMACS multisort kit according to manufacturer's instructionss (Miltenyi Biotec Inc, Sunnyvale, California). DNA was isolated usingg the QIAamp Blood Kit according to manufacturer's instructions (Qiag'en, Hilden,, Germany). Signal joint T cell receptor excision circle (TREC) numbers
weree quantified by using a real-time PCR method as previously described
34,35 5
Results s
MPAMPA inhibits T cell proliferation but not Ki67 expression
TT cell proliferation after stimulation of diluted whole blood of patients treated withh MMF in addition to HAART, assessed by thymidine incorporation, was almostt completely inhibited. This inhibitory effect on T cell proliferation was completelyy reversed when treatment with MMF was discontinued (figure 1). Stimulationn of isolated cells from a patient additionally treated with MMF in in vitrovitro cultures revealed dose dependent inhibition of T cell proliferation (measuredd by thymidine incorporation) by his own plasma (data not shown). Thesee data suggest a clear effect of MPA on proliferation in vivo. However, ex vivovivo Ki67 expression by naïve, memory and effector CD4+ and CD8+ T cells wass not further decreased by additional treatment with MMF compared to HAARTT alone (figure 2a and 2b). In order to study this apparent discrepancy wee looked at the effect of MPA on thymidine incorporation, CFSE dilution and Ki677 expression by T cells in vitro. A differential effect of MPA on thymidine incorporationn and CFSE dilution as compared to Ki67 expression after stimulationn in vitro was observed. MPA treatment was indeed able to inhibit thymidinee incorporation for at least 8 days (figure 3) and to inhibit CFSE dilutionn at least up to 6 days of stimulation (data not shown). Ki67 expression inn the presence of MPA however was elevated at day 4, its expression being evenn more pronounced at day 6 (figure 3). Since thymidine incorporation was stilll inhibited 4 days after the elevation of Ki67 expressing T cells this excludedd the possibility that MPA had lost its inhibitory effect with time of culture.. Therefore these experiments confirm that Ki67 expression occurs at ann early step of the cell cycle (G1 phase) x that is not inhibited by MPA. Howeverr the percentage of Ki67 expressing cells does appear to be decreasedd in stimulated cells treated with MPA, which is probably due to the lackk of Ki67 expressing progeny of MPA treated cells.
Figuree 1 Median percentage of control thymidine incorporation in whole blood
culturess corrected for T cell number
acutee HIV-1 infection chronicc HIV-1 infection
H A A R T / / MMF+1 1 HAART-i) )
MMF--1 MMF--1
M A N 003 M A NN 004 M A NN Oil M A NN 001 M A NN 005 M A NN 009 M A NN 012 H A A R T - / / M M F + 1 1 HAART-+I IMMF--1 MMF--1
-- M A N 008 "" M A N 002 NN 014 M A N 006 M A N 007 - M A NN 013 NN 010Leftt panel; patients with acute HIV-1 infection during treatment with HAART. Right panel; patientss with chronic HIV-1 infection during treatment with HAART. Median percentage of thymidinee incorporation of patients during additional treatment with MMF are compared to medianss after discontinuing MMF treatment (grey symbols) and medians of patients not treatedd with MMF (white symbols).
StoppingStopping MMF treatment caused a transient increase in Ki67 expressing T cellscells and apoptosis
Inn order to study the effect of MPA on Ki67 expression at an individual level, wee continued to follow patients when treatment with MMF was discontinued. Oncee treatment with MMF was withdrawn a transient increase was found in apoptosiss (data not shown) and Ki67 expression within CD4+ and CD8+ T cells inn all but one individual (figure 2 right panels).
Figuree 2 Ex vivo Ki67 expression by CD4+ and CD8+ T cells
acutee HIV-1 infection AA 30
B B
00 10 20 30 40 50 60 70 80 90 100 chronicc HIV-1 infection
00 5 10 20 30
00 10 20 30 40 50 60 70 80 90 100 limee on therapy
(weeks) ) acutee HIV-1 infection
00 5 10 20 30 timee after discontinuing MMF
(weeks) )
00 10 20 30 40 50 60 70 80 90 100 00 5 10 20 30
time e (weeks) )
timee after discontinuing MMF (weeks) ) HAART+C C M M F + j j HAART+J J MMF-- 1 ' M A NN 003 MAN 004 -- MAN 011 -- MAN 001 - M A NN 005 -- MAN 009 -- MAN 012 HAART+rr -M -M F + jj HAART+[ [ MMF F rfF-rfF- "j - M A NN 008 - M A NN 002 - M A NN 014 "MANN 006 - M A NN 007 - M A NN 013 - M A NN 010 HAART+| | MMF--TT
A A
HAART+J J MMF---- MAN 003 -- MAN 004 -- MAN 011 - M A NN 001 -- MAN 005 - M A NN 009 -- MAN 012 HAART+i i MMF+1 1 HAART+| | MMF--- M A NN 008 "MANN 002 - M A NN 014 - M A NN 006 - M A NN 007 - M A NN 013 - M A NN 010Inn the left panels patients treated with MMF (grey symbols) are compared to patients treated withh HAART alone (white symbols). Right panels illustrate Ki67 expression when treatment withh MMF is discontinued, time point zero being the last time point indicated in the left panel. AA Percentage Ki67 expression within total CD4+ T cells in acute (upper panel) and chronic
(lowerr panel) HIV-1 infection during treatment. B Percentage Ki67 expression within total CD8++ T cells in acute (upper panel) and chronic (lower panel) HIV-1 infection during
Figuree 3 Representative in vitro experiment comparing thymidine incorporation with
Ki677 expression in CD8+ T cells after stimulation with aCD3 and aCD28
Q Q SS 2 0 -Ki67 7 expression n Thymidine e incorporation n medium m aCD33 + aCD28 aCD33 + aCD28 + MPA medium m
aCD33 + aCD28 aCD33 + aCD28 + MPA
-- 30000 -- 25000 •=• 20000 0 -- 15000 -- 10000 5000 0
Thymidinee incorporation (right axes) in stimulated MPA treated cells (grey dots) is compared too stimulated control cells (white dots) and unstimulated control cells (black dots) up to 8 days off culture. After 4 and 6 days of culture Ki67 expression in CD8+ T cells (left axes) in MPA treatedd cells (grey bars) is compared to unstimulated control cells (black bars) and stimulated controll cells (white bars).
NoNo detrimental lymphocyte reconstitution during treatment with MMF
Lymphocytee reconstitution during treatment with HAART is generally biphasic withh an initial steep rise in CD4+ T cell count during the first 3 weeks of treatment,, reflecting redistribution from the lymphoid tissues, followed by a moree gradual increase 37. Given that MPA inhibits lymphocyte proliferation, MPAA might hamper lymphocyte reconstitution or could even reduce lymphocytee counts. Therefore we compared the kinetics of total and naïve CD4++ T cell counts. To consider the effects of redistribution and to account for fluctuations,, pre-treatment levels were compared to mean counts of week 4, 8
andd 12 and to mean counts of week 48, 56 and 64 during treatment. Additionall treatment with MMF had no effect on the increase in total CD4+ T celll numbers over time (figure 4). Even though variability between patients wass high, reconstitution of naïve CD4+ T cell numbers was also not affected byy MMF treatment. Furthermore, the number of TRECs in CD4+ T cells was stablee during treatment and did not change by additional treatment with MMF (dataa not shown).
Figuree 4 Total CD4+ T cell counts (per pi blood) pre-treatment compared to the
meann of weeks 4/8/12 and weeks 48/56/64 during treatment
acutee HIV-1 infection chronicc HIV-1 infection
Q Q D D HAART+f f M M F + i i HAART+I I MMF--
1 1
MANN 003 MANN 004 MANN Oil MANN 001 MANN 005 MANN 009 MANN 012 HAART+T T M M F + j j HAART+I I MMF-- i MANN 008 MANN 002 MANN 014 MANN 006 MANN 007 MANN 013 MANN 010Patientss with additional MMF treatment (grey symbols) were compared to patients treated withh HAART alone (white symbols) and to healthy controls (5th to 95th and 1s t to 99lh percentiles).. Left panel; patients with acute HIV-1 infection. Right panel; patients with chronic HIV-11 infection.
NoNo evidence for negative effects ofMPA on HIV-1 specific immunity
Inhibitionn of T cell proliferation might dampen the specific T cell response againstt HIV-1. To monitor a potential negative effect of MPA on HIV-1 specific
TT cell reactivity, we longitudinally measured the proportion of CD4+ and CD8+ TT cells that produce IFNy after stimulation with HIV-1 specific antigens. We weree unable to look at HIV-1 specific immunity within patient MAN009 (acute infectionn without MMF) and patient MAN010 (chronic infection without MMF) sincee they both had HLA-types that did not match the specific peptides available.. Before initiating therapy HIV-1 specific IFNy producing CD4+ lymphocytess could be detected in 3 out of 6 patients within the MMF arm and inn 5 out of 6 patients treated with HAART alone. HIV-1 specific IFNy producing CD8++ lymphocytes prior to therapy were present in 3 out of 6 patients treated withh MMF and 4 out of 6 patients within the HAART alone arm. Remarkably, HIV-11 specific IFNy producing CD4+ T cells (1 patient) and CD8+ T cells (2 patients)) were detected during MMF treatment in patients in whom no responsee could be measured before therapy. In those patients in whom a responsee could be measured before therapy was initiated a response could stilll be detected throughout treatment with MMF. Although there was a high variabilityy of IFNy production in response to therapy between all patients, at leastt no obvious decreases in the percentage of IFNy producing cells could be detectedd in patients additionally treated with MMF compared to patients treatedd with HAART alone (data not shown).
Discussion n
Heree we report the immunological effects of the immunosuppressive drug MMF,, which interferes with de novo guanine synthesis and HIV-1 replication, duringg antiretroviral treatment in HIV-1 infected patients. In these patients no significantt additional effects of MMF were observed compared to HAART alonee on HIV-1 virological parameters 26. Profound inhibition of T cell proliferationn in cultures of diluted whole blood of patients treated with MMF, as welll as in vitro inhibition of lymphocyte proliferation by plasma from a patient treatedd with MMF, suggested a strong in vivo effect of MPA. However, ex vivo Ki677 expression in the different T cell subsets appeared not to be lower when MMFF was administered concomitantly with HAART. This apparent discrepancyy can in part be explained by our in vitro studies, which showed thatt Ki67 is expressed at a step of the cell cycle prior to the inhibition by MPA.
Thuss measuring Ki67 in MMF treated individuals gives an overestimation of thee number of cells that will actually finish their cycle. However, since proliferatingg cells clonally expand in response to antigen and Ki67 is expressedd in every stage of the cell cycle of proliferating cells x one can envisagee that MPA will at least cause a decrease in the percentage of Ki67 expressingg cells (as seen in the in vitro experiments). Indeed in the clinical triall with MMF reported by Chapuis et al. 25, a minor decrease in the fraction off proliferating T cells was found. At first glance, these results seem inconsistentt with our data, which do not show a decrease in Ki67 expressing lymphocytess in patients additionally treated with MMF. The considerable effect off potent HAART alone could mask an effect of MPA, since in our study designn a five drug regimen was used as opposed to only two drugs in the studyy reported by Chapuis et al. High variability between the persons in this smalll study group could also conceal an effect of MPA and might explain the discrepancy.. Chapuis et al. determined the additional effect of treatment with MMFF after each individual's set point during treatment with HAART alone was reached.. Instead, we monitored the patients when MMF treatment was withdrawnn to investigate the effect of MPA on Ki67 expression at an individual level.. Indeed, after discontinuation of treatment with MMF we found a temporaryy increase in Ki67 and apoptosis in 5 out of 6 patients. The only exceptionn was patient MAN004, who had exceptionally high CD4+ counts and loww Ki67 expression before treatment and who turned out to serorevert during treatmentt 38. The transient increase in the proportion of dividing cells after discontinuingg treatment with MMF may be a reflection of ongoing T cell activationn during treatment with HAART. MPA will inhibit full cycle proliferation byy these cells. On a daily basis this supposedly is a small proportion of T cells inn a HAART treated person, but since the lifespan of short-lived activated memory/effectorr cells is estimated to be 2-3 weeks 39, they may still accumulatee to a measurable number in a person additionally treated with MMF.. When treatment with MMF is discontinued these proliferation-arrested cellss will start to cycle in a rather synchronized way, causing a temporary elevationn in the percentage of Ki67 expressing T cells. Interestingly, a similar transientt increase was noticed for the number of latently HIV-1 infected
restingg HLA-DR" CD4+ T cells in these patients 26. In accordance, after discontinuingg treatment with MMF an increase in apoptosis was found, which probablyy reflects activation induced cell death of the proliferating cells.
Importantly,, MMF treatment did not seem to have a negative effect on HIV-1 specificc cellular immunity. However, since the number of HIV-1 specific IFNy producingg cells is measured after in vitro stimulation of PBMCs, and thus in thee absence of MPA, it remains hard to translate these results to the in vivo situationn in the presence of MPA, especially since in vitro cultures have shown thatt MPA inhibits IFNy production by stimulated PBMCs A0A\ Still, at least HIV-11 specific T cells remain present during treatment with MMF.
Wee confirmed that the increase in total and naïve numbers of CD4+ T cells is nott affected by MPA in HIV-1 infected individuals 25'42-43. This may be explainedd by the fact that MPA acts differentially on thymocytes, resting and activatedd T cells and that these subsets contribute differentially to T cell numbers.. The key enzyme in the de novo pathway inhibited by MPA, IMPDH, existss in two isoforms. One isoform (type II), mainly used by activated lymphocytes,, is much more actively inhibited by MPA than the other isoform (typee I) used by resting cells 44,45. Since activated T cells are prone to die by activationn induced cell death, proliferation within this population presumably doess not contribute to maintain the T cell pool. Furthermore, the de novo pathwayy might be of less importance for thymocytes, since enzymes of the salvagee pathway are more active or present in higher levels in thymocytes comparedd to mature T cells 46,47. Indeed, MPA has been reported to have a limitedd inhibitory effect on proliferation of thymocytes in mice 48.
Thesee findings contribute to the positioning of MMF treatment in HIV-1 infectedd patients. Our data indicate that T cell proliferation is indeed inhibited inn patients treated with MMF and this did not have detrimental immunological effects.. However, the virological effects of MMF in addition to potent antiretrovirall therapy appear minimal26. MMF has been postulated for antiviral treatmentt of multi-drug resistant HIV-1 infection 42. Furthermore, since treatmentt with MMF causes minimal adverse effects, MMF might be promising forr therapy naïve patients not yet eligible for HAART. By inhibiting lymphocyte proliferationn and decreasing the amount of infected cells, the rate of CD4+ T
celll loss might be diminished, thereby delaying requirement for antiretroviral agents.. Indeed, recent studies showed that stopping HAART in the presence off MMF resulted in improved control of virus replication compared to patients withoutt NIMF49-51.
Acknowledgements s
Wee thank Dr José Borghans and Dr Kiki Tesselaar for stimulating discussions andd Marian Nievaard, research nurse, for excellent patient care. This research hass been funded by grant number 7010 from AIDS Fonds Netherlands and a private,, non-commercial foundation that wishes not to be named.
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