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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).
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
2 Annals of Internal Medicine Annals.org
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
Table—Continued
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
4 Annals of Internal Medicine Annals.org
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
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.
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.
6 Annals of Internal Medicine Annals.org
Signal-to-Cutoff Ratio
10–1 10–1
10–2 10–2
Viremic Treated Received HIV-Negative Viremic Treated Received HIV-Negative
Transplant Transplant
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.
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
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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incidence estimation. J Clin Microbiol. 2012;50:3968-76. [PMID: Schmetzer H, et al. Graft-versus-leukemia effect following hemato-
23035182] doi:10.1128/JCM.01454-12 poietic stem cell transplantation for leukemia. Front Immunol. 2017;
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Najarro KM, Cryer CG, et al. A murine viral outgrowth assay to detect 23. Lindemans CA, Chiesa R, Amrolia PJ, Rao K, Nikolajeva O, de
residual HIV type 1 in patients with undetectable viral loads. J Infect Wildt A, et al. Impact of thymoglobulin prior to pediatric unrelated
Dis. 2015;212:1387-96. [PMID: 25883388] doi:10.1093/infdis/jiv230 umbilical cord blood transplantation on immune reconstitution and
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Anguita J, et al. Haplo-cord transplantation using CD34+ cells from a .1182/blood-2013-05-502385
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Rapid quantification of the latent reservoir for HIV-1 using a viral out- .04.011
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
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-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.
Annals.org
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.
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
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.
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
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
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
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