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Annals of Internal Medicine ORIGINAL RESEARCH

Mechanisms That Contribute to a Profound Reduction of the HIV-1


Reservoir After Allogeneic Stem Cell Transplant
Maria Salgado, PhD*; Mi Kwon, MD*; Cristina Gálvez, MS; Jon Badiola, MD; Monique Nijhuis, PhD; Alessandra Bandera, MD, PhD;
Pascual Balsalobre, PhD; Pilar Miralles, MD; Ismael Buño, PhD; Carolina Martinez-Laperche, PhD; Cristina Vilaplana, PhD;
Manuel Jurado, MD, PhD; Bonaventura Clotet, MD, PhD; Annemarie Wensing, MD; Javier Martinez-Picado, PhD†; and
Jose Luis Diez-Martin, MD, PhD†; for the IciStem Consortium‡

Background: The multifactorial mechanisms associated with unit per million cells). The only participant with detectable virus
radical reductions in HIV-1 reservoirs after allogeneic hemato- received cord blood stem cells with an antithymocyte globulin–
poietic stem cell transplant (allo-HSCT), including a case of HIV containing conditioning regimen, did not develop graft-versus-
cure, are not fully understood. host disease, and had delayed complete standard chimerism in
T cells (18 months) with mixed ultrasensitive chimera. Adoptive
Objective: To investigate the mechanism of HIV-1 eradication transfer of peripheral CD4+ T cells to immunosuppressed mice
associated with allo-HSCT. resulted in no viral rebound. HIV antibody levels decreased over
Design: Nested case series within the IciStem observational time, with 1 case of seroreversion.
cohort. Limitation: Few participants.
Setting: Multicenter European study. Conclusion: Allo-HSCT resulted in a profound long-term reduc-
Participants: 6 HIV-infected, antiretroviral-treated participants tion in the HIV reservoir. Such factors as stem cell source, condi-
tioning, and a possible “graft-versus-HIV-reservoir” effect may
who survived more than 2 years after allo-HSCT with CCR5 wild-
have contributed. Understanding the mechanisms involved in
type donor cells.
HIV eradication after allo-HSCT can enable design of new cura-
Measurements: HIV DNA analysis, HIV RNA analysis, and quan- tive strategies.
titative viral outgrowth assay were performed in blood, and HIV
Primary Funding Source: The Foundation for AIDS Research.
DNA was also measured in lymph nodes, ilea, bone marrow, and
cerebrospinal fluid. A humanized mouse model was used for in Ann Intern Med. doi:10.7326/M18-0759 Annals.org
vivo detection of the replication-competent blood cell reservoir. For author affiliations, see end of text.
HIV-specific antibodies were measured in plasma. This article was published at Annals.org on 16 October 2018.
* Drs. Salgado and Kwon share first authorship of the manuscript.
Results: Analysis of the viral reservoir showed that 5 of 6 partic- † Drs. Martinez-Picado and Diez-Martin share senior authorship of the
ipants had full donor chimera in T cells within the first year after manuscript.
transplant, undetectable proviral HIV DNA in blood and tissue, ‡ For members of the IciStem Consortium, see the Appendix (available at
and undetectable replication-competent virus (<0.006 infectious Annals.org).

C ombination antiretroviral therapy (cART) is unable


to eliminate HIV-1 infection despite effective vire-
mic control. This is attributable to a persistent latent HIV
However, transplant using CCR5 wild-type donors
also leads to a greater reduction in the latent reservoir
than is obtained with any other clinical intervention (6 –
reservoir, which is responsible for rapid rebound of 9). For example, despite the delayed viral rebound af-
replication-competent virus after treatment interruption ter interruption of cART that was observed in 2 HIV-
(1). Efforts to develop an effective curative strategy are infected patients undergoing allo-HSCT from CCR5
needed to prevent long-term adverse effects of cART, wild-type donors (the “Boston patients”) (10), these
improve patients' quality of life, and eradicate the HIV cases showed that allo-HSCT by itself was able to
pandemic (2). achieve large reductions in the viral reservoir. Transplant-
Allogeneic hematopoietic stem cell transplant (allo- associated mechanisms that reduce HIV latency and
thus may play a role in eliminating the virus need to be
HSCT) has contributed to the only known case of com-
understood to allow development of less invasive strat-
plete HIV-1 eradication (in the “Berlin patient”). The un-
egies to eradicate HIV-1 infection that may be applica-
derlying biological mechanisms are not fully understood,
ble to the broader population of HIV-infected persons
although use of a donor with a homozygous mutation without hematologic disorders requiring stem cell
in the HIV coreceptor CCR5 seemed to be key to pre- transplant.
venting HIV infection of the graft (3, 4). Other contrib- The scant experience with allo-HSCT in HIV-infected
uting factors may have been the conditioning regimen, patients prevents definitive conclusions (11). To our
which destroyed some or all reservoir T cells; an immu- knowledge, the IciStem Consortium (www.icistem.org)
nologic milieu favoring T-cell activation and reactiva-
tion of latent HIV; greater effectiveness at blocking re-
activated virus spread by CCR5-mutated donor cells See also:
compared with suppressive ART; and alloreactivity that
Editorial comment . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
could have eliminated infected cells in the recipient (5).
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ORIGINAL RESEARCH Mechanisms Contributing to Reduction of HIV-1 Reservoir After ASCT

has assembled the largest and most exhaustive obser- Quantification of HIV Reservoir in Blood
vational cohort for the study of HIV reservoir dynamics HIV DNA in peripheral blood mononuclear cells or
in HIV-positive persons who have hematologic disease bulk CD4+ T cells was repeatedly measured after allo-
and have undergone allo-HSCT. Its primary objective is HSCT in each participant, as previously described (13).
to evaluate the mechanisms responsible for the dra- Residual viremia (HIV RNA) was also measured from 9
matic reduction in HIV reservoirs associated with allo- mL of plasma (3). Leukaphereses were obtained from
HSCT. all participants in order to measure the number of in-
In this study, we selected patients from the cohort fectious units in a large number of CD4+ T cells (range,
with the longest survival and follow-up (>2 years after 11 to 137 × 106 CD4+ T cells [Appendix Table, avail-
allo-HSCT). The study extends earlier reports—which in- able at Annals.org]) in accordance with previously de-
volved single or few cases— by examining 6 patients scribed protocols (3), with the detection limit set at
with HIV-1 infection who underwent allo-HSCT from 0.005 infectious unit per million cells (IUPM).
CCR5 wild-type donors and have been extensively
studied. We analyzed reductions in HIV latency and Quantification of HIV Reservoir in Anatomical
viral-specific humoral responses with respect to factors Compartments
associated with allo-HSCT in the absence of HIV resis- Per protocol, HIV was measured in tissue biopsy
tance factors, such as CCR5Δ32 mutation. specimens only in participants who had undetectable
viral reservoirs in peripheral blood. Target cells for HIV
infection were isolated from different tissues to in-
crease sensitivity for viral detection. CD45+ cells were
METHODS isolated and processed from ileal biopsy specimens us-
Participants ing the lamina propria leukocytes viral DNA assay (14).
At the time the study was designed, IciStem in- T-follicular helper CD4+ memory T cells, defined as
cluded 23 HIV-1–infected persons who had viral sup- CD3+CD4+CD45RA⫺PD1+CXCR5+, were sorted by flow
pression due to cART and high-risk hematologic dis- cytometry from lymph node biopsy specimens ob-
ease that required allo-HSCT. Thirteen died within 2 tained using fine-needle aspiration. Magnetic cell isola-
years after transplant. Seven of the remaining 10 pa- tion of CD3+ or CD4+ T-cell populations was performed
tients survived more than 2 years after transplant, 1 of in bone marrow. In all cases, isolated cells were lysed,
whom had a CCR5Δ32 donor. Therefore, the study in- and viral DNA was quantified by using droplet digital
cluded 6 participants (IciS-01 [12], IciS-03, IciS-06, IciS- PCR with 2 different sets of primers (14).
17, IciS-27, and IciS-28) who had survived more than 2 Lumbar puncture was performed to obtain 2 to 5
years after allo-HSCT with CCR5 wild-type cells, main- mL of cerebrospinal fluid (CSF), and residual viremia
tained use of cART, and achieved remission of their he- was quantified (3).
matologic disease. All participants provided informed Quantification of HIV Antibodies
consent. The observational protocol (IciStem study) was Specific HIV-1 antibodies in longitudinal plasma
approved by the institutional ethical review boards. samples were measured using a qualitative Western
blot assay (New LAV Blot I [Bio-Rad]) and the quantita-
Chimerism Analysis tive standard and low-sensitivity versions of the VITROS
In 4 participants (IciS-01, IciS-03, IciS-06, and IciS- anti–HIV-1 assay (Ortho Clinical Diagnostics) (15).
17), analyses were performed in whole bone marrow,
peripheral blood, or both. In 3 participants (IciS-01, Humanized Mouse Viral Outgrowth Assay
IciS-03, and IciS-06), T cells and myeloid cells were pu- As an in vivo measure of residual replication-
rified from peripheral blood by immunomagnetic competent reservoir cells in blood, we used a human-
means (autoMACS [Miltenyi Biotec]) using antibodies ized mouse model modified to transfer CD4+ T cells
against CD3+ and CD13/CD33+, respectively. The min- instead of total peripheral blood mononuclear cells
imum purity of isolated leukocyte subsets was 95%. In (16). All procedures were performed according to pro-
the other 2 participants (IciS-27 and IciS-28), mononu- tocol 8927, which was reviewed by the Animal Experi-
clear lymphocytes and monocytes were isolated, and mentation Ethics Committee of the University Hospital
the minimum purity was also 95%. In all participants, Germans Trias i Pujol (registered as B9900005) and ap-
conventional chimerism analysis was performed with proved by the Catalan government according to cur-
polymerase chain reaction of short tandem repeats rent national and European Union legislation on the
(STR-PCR). In IciS-01, IciS-03, and IciS-06, when conven- protection of experimental animals. Mice were super-
tional chimerism analysis (with a sensitivity of 1%) was vised daily according to a strict protocol to ensure their
complete, ultrasensitive chimerism analysis in whole welfare and were euthanized, if required, with isoflu-
peripheral blood was also performed (Mentype DIP- rane (inhalation excess). Briefly, 50 to 250 million puri-
screen and Mentype DIPquant [Biotype]), with a sensi- fied CD4+ T cells were infused in 5 mice (10 to 50 mil-
tivity of 0.01% to 0.001%, depending on the quality and lion per mouse). Whole blood samples were collected
quantity of purified DNA. Complete chimerism was de- every 2 weeks until week 12, when possible. Plasma
fined as the absence of recipient-specific allelic pat- was used for quantification of HIV RNA using the
terns detectable by STR-PCR, with the level of sensitivity m2000 Abbott platform. Whole blood was stained to
mentioned earlier. define human T-cell engraftment as the proportion of
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Mechanisms Contributing to Reduction of HIV-1 Reservoir After ASCT ORIGINAL RESEARCH

Table. Clinical, Hematologic, and Virologic Characteristics of the 6 Patients

Characteristic IciS-01 IciS-03 IciS-06 IciS-17 IciS-27 IciS-28


Clinical characteristics
Sex Male Male Male Male Male Male
Age, y 34 51 40 46 47 44
Country of origin Spain Spain Spain Italy Spain Spain

Hematologic
characteristics
Diagnosis Burkitt NHL NK-NHL (stage IV) HL (stage IV) NHL (DLBCL) NHL (stage III) HL (stage III)
(stage IV)
Year of allo-HSCT 2012 2013 2014 2010 2013 2009
Status at transplant CR2 CR1 CR2 CR2 CR1 CR3
Donor type/graft Cord blood 7/8 HLA-identical HLA-haploidentical HLA-identical HLA-identical HLA-identical
source (mismatch in sibling/PBPC sibling/PBPC sibling/PBPC sibling/PBPC unrelated/PBPC
DRB1) +
mismatched
related
PBPC†
Donor CCR5 type CCR5 wt/wt CCR5 wt/wt CCR5 wt/wt CCR5 wt/wt CCR5 wt/wt CCR5 wt/wt
Recipient HLA A*02/02, A*25/01, B*18/15, A*02/03, B*44/51, A*03/24, B*18/51, A*01/34, B*08/18, A*26/29, B*44/49,
B*44/51, Cw12/03, Cw05/07, Cw12/14, C*07/12, C*07/16,
Cw02/05, DRB1*13/03, DRB1*07/04, DRB1*07/11, DRB1*01/11, DRB1*01/07,
DRB1*04/07, DBQ1*06/02 DQB1*02/03 DQB1*02/03 DQB1*03/05 DQB1*02/05
DQB1*03/03
Donor–recipient HLA 5/6 (DRB1)‡ 10/10 6/8 (B*44/57 and 10/10 10/10 10/10
match DRB1-07/07)
Conditioning MAC: FLU, CY, RIC: FLU and RIC: FLU, CY, and RIC: Thiotepa, FLU, RIC: FLU and CY RIC: FLU and
busulfan, and melphalan busulfan and CY melphalan
ATG
Transplant-associated Escherichia coli None Clostridium difficile EBV reactivation None CMV reactivation
infections BK virus CMV reactivation (treated with
hemorrhagic rituximab)
cystitis
GvHD prophylaxis CsA + steroids CsA + Mtx Posttransplant CY + CsA + Mtx CsA + Mtx Tacrolimus +
CsA + MMF sirolimus
GvHD No Acute: Mild (by Acute: Severe (by No Chronic: Mild (by Acute (by day 12,
month 8), month 3), month 4) grade II):
affecting the affecting the skin Affecting the skin
skin and intestines and intestines
Chronic: Moderate
Day of neutrophil 15 18 22 15 11 11
engraftment
Day of platelet 31 9 25 16 11 21
engraftment
Time to complete
chimerism, mo§
Peripheral blood 2 1 3 1 ND ND
T lymphocytes 18 1 3 ND 5.5 1
Bone marrow 12 6.5 6 ND ND ND
Immunosuppression No No No No No No
at last follow-up
Status at last Alive with CR Alive with CR Alive with CR Alive with CR Alive with CR Alive with CR
follow-up

Virologic
characteristics
Time from HIV 1 27 2 16 8 11
diagnosis to
allo-HSCT, y
Time from start of ART 1 19 2 13 8 11
to allo-HSCT, y
HIV tropism R5 Dual R5/X4 Dual R5/X4 ND ND ND
Posttransplant HIV ABC + 3TC + TDF, FTC, RAL TDF, FTC, RAL TDF–FTC + DRV/r + 1. FTC + TDF + EFV 1. FTC + TDF + RAL
ART RAL, RAL 2. ABC + 3TC + 2. ABC + 3TC +
maraviroc EFV RAL
3. ABC + 3TC + 3. ABC + 3TC +
rilpivirine DTG
Continued on following page

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ORIGINAL RESEARCH Mechanisms Contributing to Reduction of HIV-1 Reservoir After ASCT

Table—Continued

Characteristic IciS-01 IciS-03 IciS-06 IciS-17 IciS-27 IciS-28


Plasma VL before 65 <50 <50 <40 <1 <1
allo-HSCT, HIV-1
RNA copies/mL
CD4+ T-cell count 0.720 0.800 0.151 0.155 0.747 0.891
before allo-HSCT,
× 109 cells/L
CD4+ cell count 3 mo 0.410 0.558 0.324 0.320 0.160 0.390
after allo-HSCT,
× 109 cells/L
Maximum CD4+ T-cell 0.891 0.660 0.759 0.773 0.815 2.550
count after
allo-HSCT,
× 109 cells/L
Detectable plasma VL Yes㛳 No No No No No
after allo-HSCT
3TC = lamivudine; ABC = abacavir; allo-HSCT = allogeneic hematopoietic stem cell transplant; ART = antiretroviral therapy; ATG = antithymocyte
globulin; CMV = cytomegalovirus; CR = complete remission; CsA = cyclosporine A; CY = cyclophosphamide; DLBCL = diffuse large B-cell
lymphoma; DRV/r = darunavir + ritonavir; DTG = dolutegravir; EBV = Epstein–Barr virus; EFV = efavirenz; FLU = fludarabine; FTC = emtricitabine;
GvHD = graft-versus-host disease; HL = Hodgkin lymphoma; MAC = myeloablative conditioning; MMF = mycophenolate mofetil; Mtx = metho-
trexate; ND = no data; NHL = non-Hodgkin lymphoma; NK = natural killer; PBPC = peripheral blood progenitor cell; RAL = raltegravir; RIC =
reduced-intensity conditioning; TDF = tenofovir disoproxil fumarate; VL = viral load; wt = wild-type.
†Single cord blood transplant supported with third-party HLA-mismatched CD34+ cells (haplo-cord transplant).
‡ 3/10 with a third-party donor.
‡ Conventional chimerism (polymerase chain reaction of short tandem repeats).
㛳 Ultrasensitive VL with limit of detection of 1 copy/mL.

human CD45+ cells in the total lymphocyte gate and IciS-01 received a myeloablative single cord blood
activated CD4+ T cells (hCD45+CD3+CD8⫺HLADR+CD transplant supported with third-party HLA-mismatched
69+CD25+) (Appendix Figure 1, available at Annals CD34+ cells (haplo-cord HSCT) (12, 17). IciS-03, IciS-17,
.org). We also lysed blood cells and quantified HIV and IciS-27 underwent reduced-intensity, conditioned
DNA as previously described (14). Spleen samples allo-HSCT from HLA-matched related donors. IciS-06
were collected at the last time point and were mechan- received a reduced-intensity, conditioned, nonmanipu-
ically disaggregated and used to quantify HIV DNA with lated transplant from an HLA-haploidentical donor,
droplet digital PCR (14) after lysis of erythrocytes. with posttransplant cyclophosphamide for graft-versus-
Statistical Analysis host disease (GvHD) prophylaxis (18). Finally, IciS-28 re-
ceived a reduced-intensity, conditioned, HLA-matched
Given the small number of patients, no statistical
transplant from an unrelated donor.
analysis was performed.
All participants achieved complete standard chi-
Role of the Funding Source merism in peripheral blood and bone marrow in the first
This study was supported by the Foundation for 12 months after allo-HSCT (Table). IciS-01 showed de-
AIDS Research (amfAR) through the amfAR Research layed achievement of complete T-lymphocyte chimer-
Consortium on HIV Eradication (ARCHE) program ism (18 months) compared with patients with available
(grants 108930-56-RGRL, 109293-59-RGRL, and 109552- data. Data on ultrasensitive chimerism in peripheral
61-RGRL) as well as Dutch Aidsfonds grants 2013034 blood were available for IciS-01, IciS-03, and IciS-06;
and 2016026. Ms. Gálvez was supported by the PhD only IciS-01 showed mixed chimerism at the last follow-
fellowship of the Spanish Ministry of Education, Culture up. Four patients had posttransplant GvHD; 3 of them
and Sport (FPU15/03698). The funding sources had no had acute GvHD, and 2 had chronic GvHD that was
role in the design or conduct of the study or the deci- treated with immunosuppression (Table).
sion to submit the manuscript for publication.
HIV Reservoir in Blood and Anatomical
Compartments
RESULTS Comprehensive virologic studies were performed
The 6 patients selected for this study survived more in blood and tissue samples from the 6 participants
than 2 years after transplant with CCR5 wild-type donor (Figure 1 and Appendix Table). The blood HIV reser-
cells. All of them showed complete remission of their voir (proviral HIV DNA analysis and quantitative viral
hematologic disease, no longer had immunosuppres- outgrowth assay [qVOA] in blood cells and HIV RNA
sion, and maintained cART during and after transplant; analysis in plasma) was undetectable in 5 of 6 partici-
only participant IciS-06 interrupted cART from days 5 to pants at the last follow-up. Of note, cell input for both
24 due to severe mucositis, with no evidence of viral HIV DNA analysis and qVOA was similar to that in pre-
rebound. Hematologic and virologic characteristics of vious reports of allo-HSCT and substantially higher than
the patients are shown in the Table. in other studies (7, 19). Conversely, low virus levels
Different transplant strategies were used according were consistently detected in blood samples from
to the decisions of the participants' hematologists. IciS-01 (453 HIV DNA copies per million CD4+ T cells, 3
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Mechanisms Contributing to Reduction of HIV-1 Reservoir After ASCT ORIGINAL RESEARCH
HIV RNA copies per milliliter of plasma, and 0.13 IUPM HIV-Specific Humoral Response
[replication-competent virus]). We explored HIV humoral response dynamics in
HIV was also undetectable in CSF and cells from plasma samples after allo-HSCT (Appendix Figure 3,
bone marrow, lymph node, and ileal biopsy specimens available at Annals.org). All participants lost the p18
in all participants, in line with previous observations in band. We observed no other missing bands in IciS-01;
blood (Figure 1 and Appendix Table). however, IciS-03 and IciS-06 showed decreasing p31
antibody levels, and IciS-06 and IciS-17 lacked p55 and
Longitudinal Correlation of Hematologic and p24 bands. More important, we did not detect any viral
HIV Reservoir Parameters antibodies in IciS-28 by month 88, suggesting that this
Five of 6 participants had undetectable HIV reser- patient experienced seroreversion. Overall, a longer in-
voirs (Appendix Figure 2, available at Annals.org). All 5 terval after allo-HSCT seemed to be associated with
had peripheral blood progenitor cells as the graft greater antibody clearance among patients receiving
source; 4 developed GvHD; and all 5 achieved com- cART. These data were confirmed with the low-
plete chimerism in peripheral blood, bone marrow, or sensitivity VITROS analysis, which showed decreased
T lymphocytes within the first year after transplant. Con- levels of HIV antibodies and a progressive loss over
versely, the only participant with a detectable HIV res- time after allo-HSCT (Figure 3). IciS-28 also showed an-
ervoir (IciS-01) received a cord blood transplant with a tibody levels close to those of the HIV-negative donors.
conditioning regimen that contained antithymocyte Overall, the data suggest limited de novo humoral re-
globulin (ATG). This participant did not develop GvHD sponses that could sustain the HIV-specific immuno-
and had mixed chimera in T cells up to posttransplant globulin levels in the plasma of these patients.
month 18, as measured by standard methods.
Longitudinal follow-up of IciS-06 is shown in Figure Humanized Mouse VOA
2. This participant showed mixed chimerism in periph- We transferred large numbers of CD4+ T cells puri-
eral blood in the first few weeks after transplant, with fied from the participants' peripheral blood to immuno-
concomitant detection of persistent reservoirs. By suppressed mice to detect any replication-competent
month 3, the patient developed acute grade III GvHD blood cell reservoir (Figure 4, A). As a control, we also
after withdrawal of immunosuppression coinciding with transferred cells from an HIV-infected person who had
achievement of full donor chimerism. Coincidentally, not undergone transplant, was receiving long-term
residual viremia became undetectable in plasma. Cell- cART, and had a standard HIV reservoir size (1.6 IUPM)
associated HIV DNA was also undetectable at that (20).
point, showing a 2-fold reduction in just 4 months. Clin- We detected high levels of HIV RNA in the plasma
ical data suggest that similar phenomena may have oc- of humanized mice infused with control CD4+ T cells
curred in the other 4 patients given that all had full (Figure 4, B). Cell-associated HIV DNA was also de-
chimerism within 6 months after allo-HSCT and/or tected in blood and spleen cells from the same infected
GvHD (Table). mice (Figure 4, C and D). Conversely, none of the mice

Figure 1. HIV reservoirs measured in blood and tissues after transplant.

Blood Anatomical Compartments


Assay HIV DNA qVOA usVL HIV DNA usVL HIV DNA HIV DNA
Tissue/cell Blood/ Blood/ Plasma Ileum/ CSF Lymph Bone
CD4+ CD4+ CD45+ Node Marrow
CD4+ Tfh CD4+

10 000 10 000
Infected Cells per 106 Cells

1000 1000
HIV-1 RNA, copies/mL

100 100

10 10

1 1

0.10 0.1

0.01 0.01

IciS-01 IciS-03 IciS-06 IciS-17 IciS-27 IciS-28

Data are from the last collected sample for each patient. Open symbols represent undetectable values (only IciS-01 had detectable values). In those
cases, the limit of detection for the sample varied on the basis of cell/volume input, and that value is represented. CSF = cerebrospinal fluid;
qVOA = quantitative viral outgrowth assay; Tfh = T-follicular helper cells; usVL = ultrasensitive viral load.

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ORIGINAL RESEARCH Mechanisms Contributing to Reduction of HIV-1 Reservoir After ASCT

Figure 2. Peripheral blood standard donor chimerism, proviral HIV DNA, and plasma HIV RNA evolution after transplant in
IciS-06.

Acute
GvHD
Immuno-
suppres- Immunosuppression No immunosuppression
sion
2200 HIV DNA (copies/106 PBMCs)
2100 HIV RNA (copies/mL)
HIV RNA or DNA Level

Donor cells
50
100

Donor Cells, %
40
80
30
60
20

10 40

0 20

0
–2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Month

Open diamonds and circles indicate undetectable HIV RNA (ultrasensitive viral load) and proviral HIV DNA, respectively, and represent the limit of
detection of each technique, which is based on cell/plasma input. For chimerism expressed as percentage of donor cells, open squares indicate full
donor chimera. GvHD = graft-versus-host disease; PBMC = peripheral blood mononuclear cell.

infused with cells from the 6 allo-HSCT recipients had HIV-infected persons. The IciStem consortium provides
detectable virus in plasma or cell-associated HIV DNA an opportunity to exhaustively study HIV remission in
in the blood or spleen after 4 to 13 weeks of follow-up. multiple HIV-infected persons who have undergone
Of note, median survival of the mice was 6 weeks (in- allo-HSCT, including the 6 long-term survivors de-
terquartile range, 5 to 12 weeks). Also, the median of scribed in this article. Not only have we confirmed the
maximum engraftment of human lymphocytes in the reduction of the HIV reservoir in blood (6, 7), but 5 of 6
mice was 34% (interquartile range, 15% to 45%). participants eliminated any measurable HIV reservoir,
Among engrafted human CD4+ T cells, activation levels as determined by highly sensitive techniques (10 to
reached a median of 95% (interquartile range, 80% to 100 times more sensitive than those used in previous
97%), suggesting optimal conditions for eventual HIV studies [21]) in lymph nodes, ilea, bone marrow, and
reactivation (Appendix Figure 4, available at Annals CSF.
.org). The only patient who had a detectable reservoir
Because IciS-01 did not show reactivation, we also underwent cord blood allo-HSCT with an ATG-
tested cells from an HIV-infected person who did not containing conditioning regimen, did not develop
undergo transplant, was receiving cART, and had a sim- GvHD, and had longer persistence of recipient cells in
ilarly small HIV reservoir (0.13 IUPM). HIV DNA (1000 the T-cell compartment. All of the other participants,
copies per million cells) was detected in the spleen and who did not have a detectable reservoir, reached full
blood of mice with human cells transferred from this donor chimerism within a year, and 4 of them devel-
person, proving the robustness of the technique. oped GvHD, although we cannot confirm that those
This model suggested that immediate viral re- events converged in time for all of them. Exhaustive
bound was not likely after discontinuation of cART in follow-up of 1 of the participants with complete viral
the 6 transplant recipients. Moreover, the virus in clearance showed that HIV became undetectable coin-
IciS-01 might have low inducibility under in vivo physi- cidentally with achievement of complete donor chime-
ologic conditions. rism and development of GvHD.
These results are in line with those of previous re-
ports, where episodes of GvHD and achievement of
DISCUSSION complete chimerism also coincided with substantial re-
Previous studies have shown that allo-HSCT can re- ductions in the viral reservoir (4, 6, 7, 9). In contrast to
sult in a significant reduction in the latent HIV reservoir the Boston patients, the IciStem participants included
(6 –9) and, in a unique case linked to transplant of in our study were all free of immunosuppression at the
CCR5-mutated cells, even eradication of the virus (4, last follow-up with T-cell immune reconstitution and
19), making HIV cure a feasible target. However, the had longer posttransplant survival. HIV-specific sero-
specific mechanisms that contributed to the decline in reversion at 8 years after transplant in IciS-28 suggests
viral reservoirs in these persons are not fully under- that longer time to remission might contribute to HIV
stood, in part due to scant experience with allo-HSCT in clearance.
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Mechanisms Contributing to Reduction of HIV-1 Reservoir After ASCT ORIGINAL RESEARCH
We postulate that replacement of recipient hema- On the other hand, whether the absence of clini-
topoietic cells with donor cells (that is, achievement of cally significant GvHD in this setting also played a role
complete chimerism in all compartments, with subse- is difficult to determine with certainty. Clinically evident
quent exertion of alloreactivity by the healthy donor im- GvHD is one of the manifestations of alloreactivity when
mune system) might be a major factor in HIV remission graft-versus-host immune responses target recipient
after allo-HSCT in the setting of CCR5 wild-type donor tissues other than hematopoietic cells, and it occurs fre-
transplantation, as previously suggested (7). After allo- quently after allo-HSCT. Of note, the ability of cord
HSCT, graft-versus-host immune responses against allelic blood cells to exert potent antitumor activity has also
variants of major and minor histocompatibility complex been observed with low rates of GvHD (24). However, the
molecules contribute to a graft-versus-leukemia effect long-term persistence of recipient cells in the T-cell com-
that is the basis of the therapeutic effect of allo-HSCT partment in IciS-01 clearly contrasts with the achievement
on hematologic disease (22). Similarly, this potent allo- of complete chimerism in the other participants. Evalua-
reactive immune effect may contribute to eradication of tion of additional persons without GvHD is needed to bet-
latently HIV-infected recipient cells through a “graft- ter understand the role of GvHD among the other poten-
versus-HIV-reservoir” effect. tial factors associated with allogeneic transplant in the
Specific transplant-associated characteristics may eradication of HIV.
explain why HIV persisted in IciS-01. For example, the Finally, the specific posttransplant immunosup-
immunosuppressive effect of ATG combined with use pressive regimen may also play a role in this setting.
of a less mature graft source (cord blood cells) may have After infusion of donor hematopoietic cells, posttrans-
moderated the potential graft-versus-HIV-reservoir effect. plant immunosuppression exerts its inhibitory effects
Delayed immune T-cell reconstitution is the primary mainly in donor immune cells to prevent severe GvHD.
drawback of cord blood transplants because of the im- This could have a dual effect in the setting of HIV infec-
mature nature of the engrafted cells. In vivo profound tion depending on the mechanism of action, dose, and
T-cell depletion resulting from ATG-containing regi- timing. On one hand, it could prevent or modulate al-
mens further intensifies long-lasting impairment of im- loreactivity against the residual HIV reservoir; on the
mune reconstitution. Better T-cell recovery with lower other hand, it could also limit uninfected T-cell permis-
incidence of virus reactivation and death from viral in- siveness for HIV replication, thus maximizing reservoir
fection has been reported in cord blood transplant re- reduction. Posttransplant high-dose cyclophosphamide
cipients not receiving ATG (23). (participant IciS-06), which is commonly used for GvHD

Figure 3. HIV-specific antibody determination using the VITROS enzyme immunoassay.

A VITROS Enzyme Immunoassay B Low-Sensitivity VITROS Enzyme Immunoassay


IciS-01
103 103 IciS-03
IciS-06
Signal-to-Cutoff Ratio

Signal-to-Cutoff Ratio

102 102 IciS-17


IciS-27
IciS-28
101 101 Viremic
Treated
100 100

10–1 10–1

10–2 10–2
Viremic Treated Received HIV-Negative Viremic Treated Received HIV-Negative
Transplant Transplant

C VITROS Enzyme Immunoassay D Low-Sensitivity VITROS Enzyme Immunoassay


100 20 IciS-01
IciS-03
Signal-to-Cutoff Ratio

Signal-to-Cutoff Ratio

IciS-06
IciS-17
10 IciS-27
50 IciS-28

0
0
–10 0 10 20 30 40 50 60 70 80 90 100 –10 0 10 20 30 40 50 60 70 80 90 100
Posttransplant Month Posttransplant Month

A. Absolute antibody quantification in the last sample from each transplant recipient compared with viremic and treated HIV-positive patients and
HIV-negative donors. B. Detuned low-sensitivity antibody quantification in the last sample from each transplant recipient compared with viremic and
treated HIV-positive patients and HIV-negative donors. C. Absolute antibody quantification in longitudinal plasma samples from all included
patients. D. Detuned low-sensitivity antibody quantification in all included patients.

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ORIGINAL RESEARCH Mechanisms Contributing to Reduction of HIV-1 Reservoir After ASCT

Figure 4. In vivo mouse viral rebound model.

A. In Vivo Mouse Viral Rebound Model 10 to 50 million CD4+ T cells infused


per mouse
6 patients receiving cART who had allo-HSCT with (Maximum of 250 million cells per patient) Measurements:
CCR5 wt donor cells >2 y prior Viral reactivation in mouse plasma (HIV RNA)
1 control patient receiving cART who had not Patient's cellular engraftment (proportion of human
undergone allo-HSCT CD45+ cells)
Patient's CD4+ T-cell activation (proportion of CD25+
CD69+HLADR+)
Viral reservoir in blood (HIV DNA measured with ddPCR)
Viral reservoir in spleen cells (HIV DNA measured with
5 NOD-Seid IL2gR–/–mice per patient
ddPCR)

B. HIV Viremia in Mouse Plasma C. Proviral Infection in Mouse Blood Cells D. Proviral Infection in Mouse Spleen Cells
Isolated After Euthanasia
Control
109 109 108 IciS-01
HIV DNA, log copies per

HIV DNA, log copies per


Log Copies/ml plasma

7
108 108 10 IciS-03
107 107 106 IciS-06
106 105 IciS-17
106 cells

106 cells
106
105 104 IciS-27
105
104 103
104 IciS-28
103 102
103 102 101
102 101 100
101 100 10–1
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 Control IciS- IciS- IciS- IciS- IciS- IciS-
01 03 06 17 27 28
Week Week

All 6 patients had undetectable values. Limit of detection relative to plasma volume input is shown. Error bars represent medians and interquartile
ranges of the values from the 5 mice used for each patient. allo-HSCT = allogeneic hematopoietic stem cell transplant; cART = combination
antiretroviral therapy; ddPCR = droplet digital polymerase chain reaction; wt = wild-type.

prophylaxis in unmanipulated haploidentical donor enhanced virus reactivation in these cultures over phys-
transplantation, eliminates rapidly proliferating allore- iologic conditions in mice. It seems reasonable that in
active T cells of both donor and recipient origin and the absence of cART, IciS-01 would have a longer HIV
preserves resting memory T cells, which results in effec- reactivation period than expected because this partici-
tive prevention of GvHD; this represents a potent graft- pant harbored a small replication-competent reservoir.
versus-leukemia effect together with relatively rapid im- Although we cannot rule out later HIV rebound after
mune reconstitution (25). Whether this strategy or other interruption of cART for any of the participants because
classic approaches could also enhance reservoir reduc- of undetectable reservoir levels (as happened in previ-
tion deserves further investigation. Our series prevents ous reported cases [6 –9]), our data contribute to mim-
definitive conclusions given the limited number of pa- icking of physiologic HIV reactivation dynamics.
tients and their differing GvHD prophylaxis schemes. Allo-HSCT is indicated for only a small subset of
However, the fact that all participants had discontinued HIV-1–infected persons with underlying hematologic
immunosuppressive therapy and 5 had an undetect- disease because of the high morbidity and mortality
able HIV reservoir at their last assessment suggests a associated with the procedure. In those who survive in
positive effect of immunosuppression withdrawal in
the long term, exhaustive consecutive studies using
preserving alloreactivity against both the underlying
highly sensitive techniques could provide important in-
hematologic disease and the HIV reservoir. The inde-
formation for better design of efficient, less toxic HIV
pendent contribution of these factors is difficult to eval-
cure strategies that could apply to the broader HIV-
uate in the present study, but the results suggest a mul-
infected population.
tifactorial interaction that promotes HIV remission.
We used an in vivo humanized mouse model that In conclusion, our study shows that allo-HSCT
has proved highly sensitive in detecting replication- yielded a profound long-term reduction in the HIV res-
competent reservoir in HIV elite controllers (16). Infu- ervoir, including 1 case of seroreversion, in the CCR5
sion of CD4+ T cells from an HIV-infected control pa- wild-type donor setting. Several transplant-associated
tient who had not undergone transplant led to virus factors may have contributed to this reduction. Further
reactivation in the plasma within 2 weeks, similar to studies are needed to confirm that a graft-versus-HIV-
what has been described elsewhere (26). In contrast, reservoir effect might be key to achieving a sterilizing
virus reactivation was not observed in mice infused with cure after allo-HSCT in HIV-infected persons. Detailed
cells from participants who underwent allo-HSCT. This studies of chimerism dynamics at ultrasensitive levels in
included IciS-01, who had shown low but detectable different reservoir compartments could further evaluate
levels of replication-competent virus when the ultrasen- the elimination of HIV through a graft-versus-HIV-
sitive qVOA technique was used. The unusually high reservoir effect. Studies of monitored antiretroviral
numbers of CD4+ T cells included in our qVOA (16, 27) pause in selected patients will be needed to determine
with strong phytohemagglutinin-mediated stimulation whether the observed absence of viral rebound in
8 Annals of Internal Medicine Annals.org

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Mechanisms Contributing to Reduction of HIV-1 Reservoir After ASCT ORIGINAL RESEARCH
the humanized mouse model correlates with the in Current author addresses and author contributions are
vivo experience. available at Annals.org.

From IrsiCaixa AIDS Research Institute, Badalona, Spain


(M.S.); Gregorio Marañón G. University Hospital, Gregorio References
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Current Author Addresses: Drs. Salgado, Clotet, and Critical revision of the article for important intellectual con-
Martinez-Picado and Ms. Gálvez: IrsiCaixa AIDS Research In- tent: M. Salgado, M. Kwon, J. Badiola, A. Bandera, P. Balsalo-
stitute, Hospital Germans Trias i Pujol, Carretera de Canyet, bre, P. Miralles, I. Buño, C. Martinez-Laperche, B. Clotet, A.
s/n, 08916 Badalona, Barcelona, Spain. Wensing, J. Martinez-Picado, J.L. Diez-Martin.
Drs. Kwon, Balsalobre, Buño, Martinez-Laperche, and Diez- Final approval of the article: M. Salgado, M. Kwon, C. Gálvez,
Martin: Hematology Department, Gregorio Marañón G. Uni- J. Badiola, M. Nijhuis, A. Bandera, P. Balsalobre, P. Miralles, I.
versity Hospital, Gregorio Marañón Health Research Institute, Buño, C. Martinez-Laperche, C. Vilaplana, M. Jurado, B. Clo-
Dr Esquerdo 46, 28007 Madrid, Spain. tet, A. Wensing, J. Martinez-Picado, J.L. Diez-Martin.
Drs. Badiola and Jurado: Virgen de las Nieves University Hos- Provision of study materials or patients: M. Salgado, M. Kwon,
pital, Avenida de las Fuerzas Armadas, 2, 18014 Granada, J. Badiola, A. Bandera, P. Balsalobre, P. Miralles, C. Martinez-
Spain. Laperche, C. Vilaplana, M. Jurado, B. Clotet, J.L. Diez-Martin.
Drs. Nijhuis and Wensing: Department of Medical Microbiol- Obtaining of funding: M. Salgado, M. Nijhuis, P. Balsalobre, B.
ogy, University Medical Center Utrecht, Heidelberglaan 100, Clotet, A. Wensing, J. Martinez-Picado.
3584 CX Utrecht, the Netherlands. Administrative, technical, or logistic support: P. Balsalobre.
Dr. Bandera: San Gerardo Hospital, University of Milano- Collection and assembly of data: M. Salgado, M. Kwon, C.
Bicocca, Via Pergolesi, 33, 20900 Monza, Italy. Gálvez, J. Badiola, M. Nijhuis, A. Bandera, P. Balsalobre, P.
Dr. Miralles: Microbiology and Infectious Diseases Depart- Miralles, I. Buño, C. Martinez-Laperche, C. Vilaplana, M.
ment, Gregorio Marañón G. University Hospital, Gregorio Jurado, A. Wensing, J. Martinez-Picado, J.L. Diez-Martin.
Marañón Health Research Institute, Calle de Máiquez, 7,
28009 Madrid, Spain.
Dr. Vilaplana: Germans Trias i Pujol Research Institute, Can APPENDIX: MEMBERS OF THE ICISTEM
Ruti Campus, Carretera de Can Ruti, Camı́ de les Escoles, s/n, CONSORTIUM
08916 Badalona, Spain. The following members of the IciStem Consortium
contributed to the article but did not author it: Asier
Author Contributions: Conception and design: M. Salgado, Sáez-Cirión, Julian Schulze zur Wiesch, Johanna Maria
M. Kwon, M. Nijhuis, P. Balsalobre, B. Clotet, A. Wensing, J. Eberhard, Gero Hütter, Jürgen Kuball, Vanderson
Martinez-Picado, J.L. Diez-Martin. Rocha.
Analysis and interpretation of the data: M. Salgado, M. Kwon,
IciStem associated collaborators: Jorge Gayoso
C. Gálvez, M. Nijhuis, A. Bandera, P. Balsalobre, P. Miralles, I.
Buño, C. Martinez-Laperche, C. Vilaplana, B. Clotet, A. Wens-
Cruz, Antonio Muscatello, Alessandro Soria, and
ing, J. Martinez-Picado, J.L. Diez-Martin. Andrea Gori.
Drafting of the article: M. Salgado, M. Kwon, P. Balsalobre, J. IciStem management team: Judith Dalmau,
Martinez-Picado, J.L. Diez-Martin. Antoinet van Kessel, and Susanne Loth.

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Appendix Table. HIV Latent Reservoir in Blood and Tissues in All Samples Isolated From Each Patient*

Variable Blood Tissues

qVOA in HIV DNA in Ultrasensitive VL Ileum: HIV DNA Lymph Node: Bone Marrow: HIV CSF:
CD4ⴙ Cells CD4ⴙ Cells in CD45ⴙ Cells HIV DNA in Tfh Cells DNA Ultrasensitive VL

Annals of Internal Medicine


in CD4ⴙ Cells

Cell IUPM Cell Copies/ Plasma HIV RNA, Cell Copies/ Cell Copies/ Cell Copies/ CSF HIV
Input Input 106 copies/mL Input 106 Input 106 Input 106 RNA,
Cells Cells Cells Cells copies/
mL
IciS-01
Before SCT – – PBMCs 184 – – – – – – – – – –
Month 29 88 × 106 0.034 1 × 106 225 9 mL 5 – – – – – – – –
Month 45 63 × 106 0.129 1 × 106 453 9 mL 3 – – – – – – – –

IciS-03
Month 17 113 × 106 Negative 1 × 106 Negative 9 mL Negative 1.5 × 104 Negative – – 2 × 106 Negative 2.5 mL Negative
(<0.006) (<5) (<0.5) (CD4+) (<64) (CD3+) (<0.5) (<0.4)
6 6
Month 31 112 × 10 Negative 1 × 10 Negative 9 mL Negative 1 × 104 Negative – – 3.7 × 107 Negative 3 mL Negative
(<0.004) (<6) (<0.5) (<98) (<0.03) (<0.3)

IciS-06
Before SCT 63 × 106 0.130 1 × 106 2162 9 mL 3 1.4 × 104 4000 – – – – 3.5 mL Negative
(CD4+) (<0.3)
Month 15 23 × 106 Negative 1 × 106 Negative 9 mL Negative 3 × 104 Negative 3 × 103 Negative 8 × 105 Negative 3 mL Negative
(<0.031) (<9) (<0.5) (<34) (<342) (<1) (<0.3)

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Month 27 113 × 106 Negative 1 × 106 Negative 9 mL Negative 5.4 × 103 Negative 4.5 × 103 Negative 5.5 × 104 Negative 3 mL Negative
(<0.006) (<2) (<0.5) (<185) (<222) (<18) (<0.3)

IciS-17
Month 65 11 × 106 Negative 1 × 106 Negative 9 mL Negative – – – – – – – –
(<0.031) (<19) (<0.5)
6 6 5 4 5
Month 75 112 × 10 Negative 1 × 10 Negative 9 mL Negative 2 × 10 Negative 6.3 × 10 Negative 6.3 × 10 Negative 7 mL Negative
(<0.006) (<2) (<0.5) (<5) Bulk LN (<16) (<2) (<0.1)

IciS-27
Before SCT – – PBMCs 1137 – – – – – – – – – –
Month 45 137 × 106 Negative 1 × 106 Negative 9 mL Negative 3 × 104 Negative – – 3 × 105 Negative 3 mL Negative
(<0.005) (<7) (<0.5) (<33) (<3) (<0.3)

IciS-28
Month 88 137 × 106 Negative 1 × 106 Negative 9 mL Negative 2.7 × 105 Negative 2.3 × 103 Negative 2.7 × 105 Negative 3 mL Negative
(<0.005) (<8) (<0.5) (<4) (CD4+) (<435) (<4) (<0.3)
CSF = cerebrospinal fluid; IUPM = infectious unit per million cells; LN = lymph node; PBMC = peripheral blood mononuclear cell; qVOA = quantitative viral outgrowth assay; SCT = stem cell
transplant; Tfh = T-follicular helper; VL = viral load.
* Limit of detection is shown for negative values.

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Appendix Figure 1. Gating strategy to quantify engraftment of human cells (proportion of human CD45+ cells), CD4 (defined
as CD3+CD8⫺) cell activation, and the event of any CD8 or NK contamination.

250 000 Mouse CD45+


105 105
Side-Scatter Area 68.8
200 000

Mouse CD45
Lymphocytes 104 Human CD45 104
150 000 84.9 22.6

CD16
NK cells
0.030
100 000 103 103

50 000 0 0

–103 –103
0
0 50 000 150 000 250 000 –103
0 103 104 105 –103 0 103 104 105
100 000 200 000 Human CD45 CD56
Forward-Scatter Area

105 105 CD3+CD8– Activated


87.5
104 104
CD8

CD3
CD8+
103 0.98 103
CD3+CD8–
0 0
97.9
–103 –103
–103 0 103 104 105 –103 0 103 104 105
CD3 CD25/CD69/HLA-DR

NK = natural killer.

Appendix Figure 2. Relationship between latency


parameters measured in each participant and clinical
conditions of each allogeneic stem cell transplant.

Patient HIV Stem Cell Time to Full Graft-Versus-


Reservoir Donor Donor Chimera Host Disease
in
T Lymphocytes

IciS-01 Detectable Cord blood >1 y None

IciS-03 Undetectable PBPC <1 y* Acute

IciS-06 Undetectable PBPC <1 y Acute

IciS-17 Undetectable PBPC ND None

IciS-27 Undetectable PBPC <1 y Chronic


Acute and
IciS-28 Undetectable PBPC <1 y*
Chronic

ND = not determined; PBPC = peripheral blood progenitor cells.


* Full donor chimera within a month.

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Appendix Figure 3. Western blot analysis from the 6 analyzed patients.

Control Control lciS-01 lciS-03 lciS-06 IciS-17 IciS-27 IciS-28


+ – +29+45+57 +17+35 Pre +1+2+3+4+7+15+27 m+7S m+4S m+88

3 3 2 2 2 2 2 2 2 2 2 2 3 33 3 3 3
6 7 2 0 1 3 4 6 5 7 8 9 2 01 3 4 5

GP160
GP120

P68
P55
P51
GP41
P40

P31

P24

P18

lnternaI control

Gray arrows indicate bands left or reduced intensity in each case.

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Appendix Figure 4. Activation profiles of CD4 + T cells from each mouse infused with human cells from each of the studied
patients.

100 Control
Activated CD4+ T Cells, %

75

50

25

0 Mouse 1
Mouse 2
Mouse 3
0 2 4 6 8 10 12
Mouse 4
Week Mouse 5

100 IciS-01 100 IciS-03 100 IciS-06


Activated CD4+ T Cells, %

75 75 75

50 50 50

25 25 25

0 0 0

0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12
Week Week Week

100 IciS-17 100 IciS-27 100 IciS-28


Activated CD4+ T Cells, %

75 75 75

50 50 50

25 25 25

0 0 0

0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12
Week Week Week

Activation is measured as expression of HLA-DR, CD25, and CD69 together.

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