bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
1 CD32+ and PD-1+ Lymph Node CD4 T Cells Support Persistent HIV-1
2 Transcription in Treated Aviremic Individuals
3 Alessandra Notoa, Francesco A. Procopioa, Riddhima Bangaa, Madeleine Suffiottia, Jean-Marc
4 Corpatauxb, Matthias Cavassinic, Craig Fenwicka, Raphael Gottardod, Matthieu Perreaua,
5 Giuseppe Pantaleoa,e#
6
7 aService of Immunology and Allergy, Lausanne University Hospital, University of Lausanne,
8 Lausanne, Switzerland
9 bService of Vascular Surgery, Lausanne University Hospital, University of Lausanne, Lausanne,
10 Switzerland
11 cService of Infectious Diseases, Lausanne University Hospital, University of Lausanne,
12 Lausanne, Switzerland
13 dVaccine and Infectious Disease Divisions, Fred Hutchinson Cancer Research Center, Seattle,
14 Washington, USA
15 eSwiss Vaccine Research Institute, Lausanne University Hospital, University of Lausanne,
16 Lausanne, Switzerland
17 Running Head: Role of CD32 and PD-1 in Defining the HIV Reservoir
18 #Address correspondence to Giuseppe Pantaleo, [email protected]
19 Word count for the abstract: 318 Word count for the text: 4130
20
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21 ABSTRACT
22 A recent study conducted in blood has proposed CD32 as the marker identifying the ‘elusive’ HIV
23 reservoir. We have investigated the distribution of CD32+ CD4 T cells in blood and lymph nodes
24 (LNs) of healthy HIV-1 uninfected, viremic untreated and long-term treated HIV-1 infected
25 individuals and their relationship with PD-1+ CD4 T cells. The frequency of CD32+ CD4 T cells
26 was increased in viremic as compared to treated individuals in LNs and a large proportion (up to
27 50%) of CD32+ cells co-expressed PD-1 and were enriched within T follicular helper cells (Tfh)
28 cells. We next investigated the role of LN CD32+ CD4 T cells in the HIV reservoir. Total HIV
29 DNA was enriched in CD32+ and PD-1+ CD4 T cells as compared to CD32- and PD-1- cells in both
30 viremic and treated individuals but there was no difference between CD32+ and PD-1+ cells. There
31 was not enrichment of latently infected cells with inducible HIV-1 in CD32+ versus PD-1+ cells in
32 ART treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell
33 populations sorted on the basis of CD32 and PD-1 expression. CD32+PD-1+ CD4 T cells were
34 significantly enriched in cell associated HIV RNA as compared to CD32-PD-1- (average 5.2 fold
35 in treated and 86.6 fold in viremics), to CD32+PD-1- (2.2 fold in treated and 4.3 fold in viremics)
36 and to CD32-PD-1+ cell populations (2.2 fold in ART treated and 4.6 fold in viremics). Similar
37 levels of HIV-1 transcription were found in CD32+PD-1- and CD32-PD-1+ CD4 T cells.
38 Interestingly, the proportion of CD32+ and PD-1+ CD4 T cells negatively correlated with CD4 T
39 cell counts and length of therapy while positively correlated with viremia. Therefore, the
40 expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent
41 HIV-1 transcription and CD32 and PD-1 co-expression the CD4 T cell population with the highest
42 levels of HIV-1 transcription in both viremic and treated individuals.
43
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44 Importance
45 The existence of long-lived latently infected resting memory CD4 T cells represents a major
46 obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells
47 containing replication competent virus is important in order to determine the mechanisms of HIV
48 persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence
49 that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations
50 infected with HIV-1. Furthermore, CD4 T cells co-expressing CD32 and PD-1 identify a CD4 T
51 cell population with high levels of persistent HIV-1 transcription.
52
53 INTRODUCTION
54 The existence of long-lived latently HIV-1 infected resting memory CD4 T cells represents a major
55 obstacle to the eradication of HIV infection (1-6). Several studies in the recent years have greatly
56 contributed to the characterization of different CD4 T cell populations containing inducible
57 replication competent HIV-1. In the blood, central memory CD4 T cells defined by the CD45RA-
58 CCR7+CD27+ phenotype and transitional memory CD4 T cells defined by CD45RA-CCR7-CD27+
59 phenotype have been identified as major cellular compartments of the latent HIV-1 reservoir while
60 CD4 T cells with stem-cell like properties as a minor component (7, 8). Recent studies always
61 performed in blood has also shown that a series of markers such as HLA-DR, CCR6, TIGIT and
62 CD30 help to define CD4 T cell populations infected with HIV-1 (9-11). Studies performed in
63 memory CD4 T cell populations isolated from LNs of viremic and long-term treated HIV-1 infected
64 individuals have demonstrated that PD-1 positive and Tfh CD4 T cells are the major reservoir for
65 replication competent and infectious virus in both viremic and long-term treated individuals (12,
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66 13). A recent study performed in blood has proposed that the low affinity receptor for the
67 immunoglobulin G Fc fragment, CD32a, defines a small population of HIV-1 infected resting CD4
68 T cells and therefore CD32a+ CD4 T cells represent the elusive HIV reservoir (14). A recent study
69 has challenged the findings that CD32a is a marker defining the elusive HIV reservoir (15). CD32+
70 CD4 T cells expressed several markers of activation, they are not enriched in HIV DNA while they
71 are enriched for transcriptionally active HIV in long-term treated individuals. Along the same line,
72 another study performed in blood has also failed to show enrichment of HIV infected CD4 T cells
73 within CD32+ cells (16).
74 On the basis of the CD32a study and of our previous observations on the role of PD-1+/Tfh cells in
75 the HIV reservoir (12-14), we have addressed the following issues: a) the differences in the
76 percentage of CD32+ CD4 T cells in healthy versus HIV infected individuals, b) the distribution of
77 CD32 in blood versus lymphoid tissue, i.e. lymph nodes (LNs), c) the distribution of CD32 in
78 different populations of memory CD4 T cells, and d) the relationship between CD32+ and PD-1+
79 CD4 T cell populations and their role in defining the HIV reservoir.
80
81 RESULTS
82 Distribution of CD32+ CD4 T cells in blood and lymph nodes
83 Blood and LN biopsies were obtained from 9 healthy subjects, 19 long-term antiretroviral therapy
84 (ART) treated and 10 viremic individuals (Table 1). In healthy individuals, lymph node biopsies
85 were obtained from patients undergoing vascular surgery. A small percentage (<1%) of memory
86 CD4 T cells in blood expressed CD32 but there were not significant differences between healthy,
87 long-term ART treated and viremic individuals (Fig 1A and B). Memory CD4 T cells from LNs of
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88 the same individuals contained significantly greater percentages of CD32+ cells as compared to
89 blood (healthy P = 0.003, ART treated P < 0.0001, viremics P = 0.002). The percentage of LNs
90 CD32+ CD4 T cells was significantly greater in viremics versus long-term ART treated individuals
91 (Fig 1A and B) (P = 0.0003) but not versus healthy individuals (Fig 1A and B).
92 These results indicate that CD32 expression in memory CD4 T cells from blood and LNs is not
93 restricted to HIV-1 infected individuals and that CD32+ CD4 T cells are greatly enriched in LNs.
94
95 Distribution of CD32 in LN and blood memory CD4 T cell populations
96 Next, we investigated the distribution of CD32 in different memory LN CD4 T cell populations
97 defined by the expression of CXCR5 and PD-1 in healthy, long-term ART treated and viremic
98 individuals. Memory CD32+ CD4 T cells were consistently enriched in PD-1+CXCR5- and PD-
99 1+CXCR5+ CD4 T cell populations in the three study groups (P < 0.05 for all study groups) (Fig
100 2A and B). The PD-1+CXCR5+ CD4 T cells correspond to Tfh cells (12, 17-19). PD-1+ CD4 T cells
101 were greatly enriched within the CD32+ CD4 T cell population in the three study groups and 40
102 and 50% of PD-1+ CD4 T cells co-expressed PD-1 and CD32 in long-term ART treated and viremic
103 individuals, respectively (Fig 2C) (P < 0.0001 in healthy and ART treated and P = 0.002 in
104 viremics). Similarly, a substantial proportion of Tfh cells ranging between 10% in ART treated and
105 20% in viremic individuals were contained within the CD32+ CD4 T cell population (Fig 2D) (P <
106 0.0001 and P = 0.002, respectively). However, as compared to the CD32+ CD4 T cell population,
107 Tfh cells were significantly enriched within the PD-1+ CD4 T cell population (P < 0.0001) (S1
108 Fig). The distribution of memory CD4 T cell populations defined by CD32 and PD-1 was also
109 investigated in blood (S2 Fig). The CD32-PD-1- CD4 T cell population was significantly reduced
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110 in viremic individuals as compared to the healthy subjects (P = 0.0007). No differences in the
111 percentage of CD32+PD-1- and CD32+PD-1+ CD4 T cells were observed in the three study groups
112 while CD32-PD-1+ CD4 T cells significantly increased in viremic individuals (13.1% in viremics
113 versus 4% in healthy, P = 0.0007 and 6.1% in treated, P = 0.006).
114
115 Relationship between CD32+ and PD-1+ CD4 T cell populations
116 The simultaneous assessment by mass cytometry of 30 phenotypic markers defining different cell
117 lineages, memory differentiation, cell activation and cell trafficking has provided the possibility to
118 analyzed more in depth the differences between CD32+ and CD32- lymph node memory CD4 T
119 cell populations in the three study groups. The large majority (13 out of 16) of markers defining
120 chemokine receptors (CXCR3, CCR6, CCR4), cell activation (CD25, CD38, HLA-DR), memory
121 cell differentiation (ICOS, CD57, CD27, CD127, CXCR5, PD-1, CD40L) and HIV-1 co-receptors
122 (CCR5 and CXCR4) were greatly increased in CD32+ as compared to CD32- CD4 T cells
123 regardless of the study group (Fig 3A and S3 Fig). Only CCR7, CD127 and CD27 were unchanged.
124 The heat maps in Fig 3A show also the differences among the study groups. ICOS, CD38, PD-1
125 are enhanced within the CD32+ cells (P < 0.0001) while CCR4 and CD127 were slightly reduced
126 within the CD32- cells in viremic individuals. Several markers are downregulated in ART treated
127 individuals as compared even to healthy individuals.
128 Due to the overlap between CD32+ and PD-1+ CD4 T cell populations, the distribution of the same
129 panel of makers was analyzed in lymph node memory CD4 cell sub-populations defined by the
130 expression of CD32 and PD-1 in the three study groups (Fig 3B). The heat maps and the Venn
131 diagram show that the expression of PD-1 determines a clear separation between the four cell
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132 populations. It appears that PD-1-CD32- and PD-1-CD32+ CD4 T cells from one side and PD-1+
133 CD32- and PD-1+CD32+ CD4 T cells from the other side share a similar phenotype and are closely
134 related to each other (Fig 3B and C). The greater expression of markers such as CXCR5, ICOS,
135 CD57, CD40L, CD38, HLA-DR and reduced expression of CD127 and CCR7 indicates that PD-
136 1+ CD4 T cell populations are at an advanced stage of differentiation and activation. Of note, the
137 expression of the HIV-1 co-receptors, CCR5 and CXCR4, is greatly enhanced in the PD-1+ CD4
138 T cell populations (CCR5+ cells 21.8% in CD32-PD-1+ and 37% in CD32+PD-1+; CXCR4+ ce1ls
139 14.2% in CD32-PD-1+ and 18% in CD32+PD-1+). Cumulative data on the expression of the 15
140 markers in the four memory CD4 T cell populations defined by the expression of PD-1 and CD32
141 independently of the study group are shown in S4 Fig.
142 Taken together these results indicate that there is great overlap between CD32+ and PD-1+ memory
143 CD4 T cell populations in LNs of HIV-1 infected individuals. Importantly, the expression of PD-
144 1 independently of CD32 expression helps to define CD4 T cells potentially more susceptible to
145 HIV infection.
146
147 Role of PD-1+ and CD32+ memory CD4 T cell populations in the HIV Reservoir
148 Having defined the distribution and the phenotypic relationship between PD-1+ and CD32+
149 memory CD4 T cell populations in blood and LNs, we sought to investigate the role of these cell
150 populations in the HIV reservoir through determining whether there is: a) an enrichment of HIV
151 DNA containing cells in LN CD32+ and PD-1+ CD4 T cells, b) a difference in the frequency of
152 HIV DNA containing cells between LN CD32+ and PD-1+ CD4 cells in viremic and long-term
153 ART treated individuals, c) a difference in the frequency of cells containing replication competent
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154 HIV between LN CD32+ and PD-1+ CD4 T cells in long-term treated individuals, d) a difference
155 in HIV transcription between LN CD32+ and PD-1+ CD4 T cells in long-term ART treated
156 individuals. Memory CD4 T cells (CD45RA-) isolated from lymph nodes of seven long-term ART
157 treated and seven viremic individuals were sorted on the basis of CD32 and/or PD-1 expression
158 and analyzed for the presence of total HIV DNA. A caveat of this analysis is that because of the
159 overlapping between CD32+ and PD-1+ CD4 T cell populations, a proportion of cells co-expressing
160 PD-1 and CD32 are present in both sorted CD32+ and PD-1+ cell populations (S5A Fig). However,
161 because of the limited number of cells particularly in LNs of ART treated individuals, it was not
162 possible to assess the total HIV DNA in the four cell populations defined by the expression of
163 CD32 and PD-1 (CD32-PD-1-, CD32+PD-1-, CD32-PD-1+ and CD32+PD-1+). CD32+ and PD-1+
164 CD4 T cell populations were enriched in cells containing HIV DNA as compared to CD32- and
165 PD-1- cell populations. With regard to CD32+ versus CD32- the differences were significant in the
166 ART treated individuals (1.83 fold, P = 0.01) and viremics (23.7 fold, P = 0.01) (Fig 4A). With
167 regard to PD-1+ versus PD-1- CD4 T cell populations, there was only a trend toward higher HIV
168 DNA in PD-1+ versus PD-1- in ART treated individuals while the differences were significant in
169 viremic individuals (6.5 fold, P = 0.01) (Fig 4A).
170 These results show an enrichment of HIV infected cells within CD32+ and PD-1+ LN memory CD4
171 T cells but not substantial difference between these cell populations. The massive proportion of
172 cells infected on the basis of the total HIV DNA content in CD32+ and PD-1+ CD4 T cells are
173 consistent with the phenotypic data (Fig 3) showing increased expression of HIV-1 co-receptors in
174 these cell populations and indicate that they may serve as a major reservoir of HIV-1.
175 To estimate the frequencies of HIV-1 infected cells containing inducible replication competent
176 virus, a quantitative virus outgrowth assay (QVOA) was performed in three ART treated
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177 individuals. The objective of the QVOA was to determine whether the exclusion of CD32+ or PD-
178 1+ cells had a different impact on the frequency of HIV-1 infected cells with inducible virus.
179 Because of the limited number of cells isolated from ART treated individuals, it was not possible
180 to perform the QVOA on CD32+ and PD-1+ CD4 T cell populations. For the QVOA, different cell
181 concentrations (five-fold limiting dilutions: 105, 2.104 and 4.103) and multiple replicates of sorted
182 viable lymph node CD32-, PD-1- and total memory (CD45RA-) CD4 T cells (S5A Fig) were co-
183 cultured with allogeneic fresh CD8 T cell-depleted blood mononuclear cells from HIV-1 uninfected
184 individuals and frequencies were estimated at day 14 using Extreme Limiting Dilution Assay (20).
185 These analyses provide the average RNA-unit per million (RUPM) for each cell population
186 investigated. The frequencies of cells with inducible HIV RNA in CD32- and PD-1- CD4 cell
187 populations were not significantly different (Fig 4B). In order to interpret correctly these results, it
188 is important to consider that sorting out CD32+ cells there are still PD-1+ cells remaining in the
189 sorted CD32- cell population (S5A Fig). Since also PD-1+ cells contain inducible HIV, it is
190 expected the finding that there is a trend towards reduction of the frequency of cells with inducible
191 HIV and not a more dramatic reduction. Along the same line, by sorting out PD-1+ cells there are
192 still CD32+ cells remaining in the sorted PD-1- cell population which explains the partial reduction
193 found. Finally, there was a trend towards higher frequencies of cells with inducible HIV RNA in
194 the total memory CD4 T cell population as compared to CD32- and PD-1- populations (Fig 4B).
195 We have recently shown that PD-1+ CD4 T cells serve as the major CD4 T cell compartment in
196 blood and LNs for replication competent and infectious HIV-1 and for active and persistent virus
197 transcription in long-term ART treated aviremic individuals (13). We then investigated whether
198 there was a difference in HIV transcription between LN CD32+ and PD-1+ CD4 T cells in ten long-
199 term ART treated and six viremic individuals. To determine the cell compartment(s) serving as
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200 sites of active and persistent HIV-1 transcription, cell-associated HIV-1 RNA was assessed in LN
201 memory CD32-PD-1-, CD32+PD-1-, CD32-PD-1+ and CD32+PD-1+ CD4 T cell populations. The
202 gating strategy for cell sorting is shown in S5B. The results indicate that the levels of cell-
203 associated HIV-1 RNA were significantly higher in CD32+PD-1+ CD4 T cells as compared to the
204 other three cell populations (Fig 4C). CD32+PD-1+ CD4 T cells were enriched in cell-associated
205 HIV-1 RNA as compared to CD32-PD-1- (average 5.2 fold in ART treated and 86.6 fold in
206 viremics), to CD32+PD-1- (2.2 fold in ART treated and 4.3 fold in viremics) and to CD32-PD-1+
207 cell populations (average 2.2 fold in ART treated and 4.6 fold in viremics). CD32+PD-1- and CD32-
208 PD-1+ cell populations had also significantly higher levels of cell-associated HIV RNA as
209 compared to CD32-PD-1- cells (P < 0.05) while no differences were found between CD32+PD-1-
210 and CD32-PD-1+ cell populations.
211 Therefore, the co-expression of CD32 and PD-1 identifies a population with greater HIV
212 transcriptional activity within Tfh cells and the sole expression of CD32 identifies a cell population
213 with levels of transcriptional activity similar to those measured in single PD-1+ CD4 T cells.
214 Recent results (21) have suggested that the expression of CD32 on blood CD4+ T cells was
215 reflective of either the presence of doublets or partial exchange of a B cell and CD4+ T cell
216 membranes. We also have carefully addressed this issue in our flow cytometry experiments. The
217 exclusion of doublets is a mandatory step in the flow cytometry analysis of cell populations. As
218 shown in S6 Fig we have totally excluded that the expression of CD32 on lymph node CD4+ T
219 cells was the result of B-T cells doublets. In the presence of B-CD4 T cell doublets we should find
220 that the expression of CD32 in the different memory CD4 T cell populations would also be
221 associated with the expression of CD19. The doublets have been excluded in two sequential
222 analyses. Similarly, CD19+ B cells and CD8+ T cells are excluded when gating for CD4+ T cells
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223 (S6A Fig). S6B Fig clearly demonstrates that there is not CD19 expression on single CD32+ CD4
224 T cells as well as CD32+PD-1+ CD4 T cells.
225
226 CD32+ and PD-1+ CD4 T cells and markers of HIV disease activity
227 We then determined the relationship between CD32+ and PD-1+ CD4 T cells and indicators of HIV
228 disease activity and the length of suppressive ART. The percentage of CD32+ CD4 T cells
229 negatively correlated with CD4 T cell count (r = -0.58, P = 0.0008) and length of ART treatment
230 (r = -0.79, P < 0.0001) while positively correlated with the levels of viremia (r = 0.68, P < 0.0001)
231 (Fig 5A). Next, we analyzed the relationships between the levels of viremia and the length of
232 treatment and the percentage of the four LN memory CD4 T cell populations sorted on the basis of
233 the expression of CD32 and PD-1. Interestingly, the percentage of CD32-PD-1- cells negatively
234 correlated with the levels of viremia (r = -0.46, P = 0.01) while the percentages of CD32+PD-1-,
235 CD32-PD-1+ and CD32+PD-1+ cells strongly correlated with viremia (r = 0.56, P = 0.001; r = 0.46,
236 P = 0.03; r = 0.65, P = 0.0001, respectively) (Fig 5B). In contrast, the percentage of CD32-PD-1-
237 cells positively correlated with the length of ART (r = 0.6, P = 0.0004) while the percentage of the
238 other three cell populations negatively correlated with the length of ART treatment (r = -0.67, P <
239 0.0001; r = -0.56, P = 0.001; r = -0.8, P<0.0001, respectively) (Fig 5C).
240 A similar pattern was observed for PD-1+ CD4 T cells (Fig 6). The percentage of PD-1+ CD4 T
241 cells negatively correlated with CD4 T cells count (r = -0.51, P = 0.004) and length of suppressive
242 ART (r = -0.58, P = 0.0009) while positively correlated with the levels of viremia (r = 0.43, P =
243 0.01).
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244 These results indicate that the expansion of both CD32+ and PD-1+ memory CD4 T cell populations
245 in lymph nodes is driven by HIV replication.
246
247 DISCUSSION
248 The percentage (range of 0.2-0.5%) of CD32+ CD4 T cells and its intensity of expression are
249 extremely low in blood thus rendering challenging to perform in depth characterization of
250 circulating CD32+ CD4 T cells. For these reasons, we decided to study CD32+ CD4 T cells in LNs.
251 Lymphoid tissues are the major anatomic compartment for HIV replication, spreading and
252 persistence and therefore they are the ideal anatomic compartment to understand the role of CD32+
253 CD4 T cells in the HIV reservoir and their relationship with PD-1+/Tfh cells that have been shown
254 to serve as a major reservoir for HIV (13, 22-29). The percentages of CD32+ CD4 T cells were
255 similar in healthy HIV uninfected, viremic and ART treated individuals in the blood. Higher
256 percentage of CD32+ CD4 T cells were found in LNs in the three study groups and were
257 significantly expanded in viremic individuals but similar percentages were found in LNs of healthy
258 and treated individuals thus indicating that CD32 expression on CD4 T cells is not exclusive of
259 HIV infection as proposed by Descours et al (14).
260 CD32+ and PD-1+ CD4 T cell populations are tightly correlated as indicated by the large proportion
261 (up to 50%) of CD32+ CD4 T cells co-expressing PD-1. The co-expression of PD-1 is important
262 for understanding the status of activation and differentiation of CD32+ cells and their role in the
263 HIV reservoir. Indeed, the expression of PD-1 together with HLA-DR and CD38 in CD32-PD-1+
264 and CD32+PD-1+ CD4 T cells indicates that these cell populations are associated with high levels
265 of cell activation and advanced differentiation as compared to the CD32-PD-1- and CD32+PD-1-
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266 cell populations. Of note, CD32+ and PD-1+ CD4 T cells expressed higher levels of the HIV-1 co-
267 receptors CCR5 and CXCR4 thus rendering these cells a preferential target for HIV infection.
268 These data are consistent with two recent studies (15, 16) showing that CD32+ CD4 T cells express
269 higher levels of activation markers and HIV co-receptors.
270 On the basis of the quantification of the total HIV DNA and of cells with inducible replication
271 competent HIV, CD32+ and PD-1+ CD4 T cells contribute equally to the HIV reservoir in long-
272 term treated individuals and no differences were also found in viremic individuals. More
273 importantly, the analysis of cell-associated HIV RNA has demonstrated equal levels of persistent
274 HIV transcription in CD32+PD-1- and CD32-PD-1+ CD4 T cell populations and significantly higher
275 levels of transcription in the CD32+PD-1+ cells. These findings are in agreement with a recent study
276 (15) failing to confirm the conclusions of Descours et al. that CD32 defines the latent ‘elusive’
277 HIV reservoir. However, CD32, independently of PD-1, defines an additional population of
278 memory CD4 T cells with persistent HIV transcription. When co-expressed with PD-1, CD32
279 defines the CD4 T cell population with the highest levels of transcription.
280 In conclusion, the combined use of CD32 and PD-1 identify three cell populations, CD32+PD-1-,
281 CD32-PD-1+ and CD32+PD-1+, of memory CD4 T cells responsible for most of persistent HIV
282 transcription in long-term treated individuals.
283
284
285
286
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287 METERIAL AND METHODS
288 Study groups
289 Samples from 9 healthy subjects, 19 subjects on suppressive ART and from 10 viremic individuals
290 not receiving ART at the time of sampling were used in this study. None of the participants under
291 ART had detectable plasma viremia at the time of the study, as assessed by viral load measurement
292 using the Ampliprep/Cobas Taqman HIV-1 Test v 2.0 (Roche), with a detection limit of 20 HIV
293 RNA copies/ml of plasma. All participants underwent lymph node biopsy (inguinal lymph nodes)
294 and blood was collected at the same time. Subject characteristics are summarized in Table 1. These
295 studies were approved by the Institutional Review Board of the Centre Hospitalier Universitaire
296 Vaudois, and all subjects gave written informed consent.
297
298
299
300
301
302
303
304
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305 Table 1. Study cohort: clinical data.
Duration of Years of Viral load CD4 count Patient ID Age Sex HIV infection suppressive ART Status (copies/ml) (cells/l) (years) ART
MP106 37 M 0.1 0 160'000 501 Treatment Naive
MP140 23 M 0.08 0 360'000 427 Treatment Naive
MP119 23 M 1.3 0 13'000 498 Treatment Naive
MP124 46 F 0.06 0 510'000 468 Treatment Naive
MP113 33 M 31.98 0 640 594 Treatment Naive
MP117 51 M 5.42 0 54'000 504 Treatment Naive
MP125 35 M 0.16 0 17'000 511 Treatment Naive
MP118 29 M 0.06 0 14'000 538 Treatment Naive
SA150 53 M 10 0 6'900 578 Treatment Naive
SA143 37 M 0.3 0 25’000 704 Treatment Naive
MP129 44 M 14.85 14.71 <20 928 ART Treated
MP068-1 45 M 3.33 3 <20 928 ART Treated
MP136 44 M 7.5 6 <20 732 ART Treated
MP123 61 M 9.25 9 <20 1270 ART Treated
MP137 39 M 3.59 2.84 24 700 ART Treated
MP064 36 M 7.88 4.49 <20 719 ART Treated
MP141 31 M 11.56 1.5 <20 558 ART Treated
MP094 47 M 8 8 <20 451 ART Treated
MP058 46 F 10.86 10 <20 666 ART Treated
MP082 53 M 22.45 8 <20 1236 ART Treated
MP096 54 F 18 13 <20 1253 ART Treated
MP103 41 M 1.62 1.16 <20 217 ART Treated
MP068-2 47 M 5.12 5 <20 487 ART Treated
MP060 42 M 21 8 <20 786 ART Treated
MP067 41 M 14.01 14 <20 609 ART Treated
MP072 47 M 18 2 <20 614 ART Treated
MP070 55 M 7.06 2 <20 615 ART Treated
MP100 36 F 13.06 10 <20 643 ART Treated
MP145 39 M 8.35 4 <20 820 ART Treated
306
15 bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
307 Isolation of lymph node cells
308 Lymph node mononuclear cells were isolated by mechanical disruption (22) and cells were
309 cryopreserved in liquid nitrogen.
310
311 Antibodies
312 The following antibodies were used: APC-H7-conjugated anti-CD3 (clone SK7, BD), PB-
313 conjugated anti-CD19 (clone HIB19, Biolegend), PB-conjugated CD8 (clone RPA-T8, BD) FITC
314 conjugated anti-CXCR5 (clone RF8B2, BD), PE-Cy7-conjugated anti-PD-1 (clone EH12.1, BD),
315 Alexa700 anti-CD4 (clone RPA-T4, Biolegend), APC-conjugated anti-CD32 (FUN-2, Biolegend),
316 ECD-conjugated anti-CD45RA (clone 2H4, Beckman Coulter). Aqua LIVE/DEAD stain kit was
317 used to determine the viability of cells. Cells were acquired on an LSRII flow cytometer using the
318 FACSDiva software (BD) and analyzed using FlowJo 9.7 (TreeStar Inc) or sorted using FACSAria
319 (BD). The CD32 antibody used in the present study is the same used in the study by Descours et
320 al. and this antibody does not discriminate between CD32a and CD32b.
321
322 CyTOF marker labeling and detection
323 Cryopreserved lymph node mononuclear cells were thawed and resuspended in complete RPMI
324 medium (Gibco; Life Technologies; 10% heat-inactivated FBS (Institut de Biotechnologies
325 Jacques Boy), 100 IU/ml penicillin, and 100 µg/ml streptomycin (BioConcept)) at 1x106 cells/ml.
326 Viability of cells in 500 μl of PBS was assessed by incubation with 50 μM of cisplatin (Sigma-
327 Aldrich) for 5 min at room temperature (RT) and quenched with 500 μl of fetal bovine serum. Next,
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328 cells were incubated for 20 minutes at 4°C with anti-CD32 APC antibody. Next, cells were washed
329 and incubated for 30 min at RT with a 50 μl cocktail of cell surface metal conjugated antibodies
330 (S1 Table). Cells were washed and fixed for 10 min at RT with 2.4% PFA. Total cells were
331 identified by DNA intercalation (1 μM Cell-ID Intercalator, Fluidigm/DVS Science) in 2% PFA at
332 4 °C overnight. Labeled samples were assessed by the CyTOF1 instrument that was upgraded to
333 CyTOF2 (Fluidigm) using a flow rate of 0.045 ml/min. FCS files were normalized to the EQ Four
334 Element Calibration Beads using the CyTOF software. For conventional cytometric analysis of
335 memory CD4 T cell populations, FCS files were imported into Cytobank Data Analysis Software.
336
337 Quantification of total HIV DNA
338 CD32+, CD32-, PD-1+ and PD-1- memory (CD45RA-) CD4 T cell populations were sorted using
339 FACSAria and lysates were directly used in a nested PCR to quantify both total HIV DNA and
340 CD3 gene copy numbers, as previously described (7).
341
342 Quantification of cell-associated RNA
343 Cell-associated HIV-1 RNA from individual samples was extracted from CD4 T cell populations
344 sorted on the basis of CD32 and PD-1 expression (CD32-PD-1-, CD32+PD-1-, CD32-PD-1+,
345 CD32+PD-1+) and subjected to DNase treatment (RNAqueous-4PCR Kit, Ambion). RNA standard
346 curves were generated after isolation and quantification of viral RNA from supernatant of ACH2
347 culture as previously described (30). One-step cDNA synthesis and pre-amplification were
348 performed as previously described (31).
349
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350 Viral outgrowth assay (VOA)
351 Different cell concentrations (five-fold limiting dilutions: 105, 2.104 and 4.103 for lymph node CD4
352 T cells) and multiple replicates of sorted viable LN total memory (CD45RA-), CD32- and PD-1-
353 memory CD4 T cells isolated from three ART treated HIV-1 infected individuals were cultured
354 with allogenic fresh CD8-depleted blood mononuclear cells (106 cells/mL) from HIV-1 uninfected
355 subjects in the presence of anti-CD3/anti-CD28 MAb coated plates (10 µg/mL) for 3 days. Cells
356 were carefully transferred to new uncoated plates post 3 days of activation. All cell conditions were
357 cultured in complete RPMI for 14 days. Medium was replaced at day 5. Supernatants were
358 collected at day 14. The presence of HIV-1 RNA was assessed by COBAS® AmpliPrep/TaqMan®
359 HIV-1 Test (Roche; Switzerland). Wells with detectable HIV-1 RNA (≥20 HIV-1 RNA copies/ml)
360 were referred to as HIV-1 RNA-positive wells. RUPM frequencies were estimated by conventional
361 limiting dilution methods using Extreme Limiting Dilution analysis
362 (http://bioinf.wehi.edu.au/software/elda/) (32, 33).
363
364 Statistical analysis
365 We performed two-tailed Mann-Whitney test to compare frequency of CD32+ CD4 T cells among
366 HIV-1 uninfected, HIV-1 infected ART treated and viremic individuals. Wilcoxon signed-rank test
367 was used to compare different subsets. Correlative analysis were done using Spearman test. These
368 analyses were done using Prism 7.0.
369 Statistical analyses comparing cell surface marker expressions (non-transformed) (Fig 3A and B)
370 in CD32+ CD4 T cells and subsets defined by CD32 and PD-1 (i.e. CD32 vs. PD-1 quadrants) were
371 assessed using linear mixed-effect models on the frequency of positive cells for each marker
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372 accounting for differences between patient groups (HIV-1 uninfected, HIV-1 infected ART treated
373 and viremic individuals) with patient-level random intercepts. P-values were adjusted using
374 Bonferroni correction and data analysis was performed using R statistical software and the lmer
375 package.
376
377 Data Availability
378 All relevant data are within the paper and its Supplemental Material files.
379
380 ACKNOWLEGEMENTS
381 This work was supported by a grant of the Swiss National Fund. The funders had no role in study
382 design, data collection and interpretation, or the decision to submit the work for publication.
383 The authors declare no competing financial interests.
384 We are grateful to Alex Farina, Navina Rajah, Line Leuenberger and Manon Geiser for technical
385 assistance.
386
387
388
389
390
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438 14. Descours B, Petitjean G, Lopez-Zaragoza JL, Bruel T, Raffel R, Psomas C, Reynes J, Lacabaratz 439 C, Levy Y, Schwartz O, Lelievre JD, Benkirane M. 2017. CD32a is a marker of a CD4 T-cell HIV 440 reservoir harbouring replication-competent proviruses. Nature 543:564-567. 441 15. Abdel-Mohsen M, Kuri-Cervantes L, Grau-Exposito J, Spivak AM, Nell RA, Tomescu C, Vadrevu 442 SK, Giron LB, Serra-Peinado C, Genesca M, Castellvi J, Wu G, Del Rio Estrada PM, Gonzalez- 443 Navarro M, Lynn K, King CT, Vemula S, Cox K, Wan Y, Li Q, Mounzer K, Kostman J, Frank I, 444 Paiardini M, Hazuda D, Reyes-Teran G, Richman D, Howell B, Tebas P, Martinez-Picado J, 445 Planelles V, Buzon MJ, Betts MR, Montaner LJ. 2018. CD32 is expressed on cells with 446 transcriptionally active HIV but does not enrich for HIV DNA in resting T cells. Sci Transl Med 447 10. 448 16. Martin GE, Pace M, Thornhill JP, Phetsouphanh C, Meyerowitz J, Gossez M, Brown H, Olejniczak 449 N, Lwanga J, Ramjee G, Kaleebu P, Porter K, Willberg CB, Klenerman P, Nwokolo N, Fox J, Fidler 450 S, Frater J. 2018. CD32-Expressing CD4 T Cells Are Phenotypically Diverse and Can Contain 451 Proviral HIV DNA. Frontiers in Immunology 9. 452 17. Kim CH, Rott LS, Clark-Lewis I, Campbell DJ, Wu L, Butcher EC. 2001. Subspecialization of 453 CXCR5+ T cells: B helper activity is focused in a germinal center-localized subset of CXCR5+ T 454 cells. J Exp Med 193:1373-81. 455 18. Breitfeld D, Ohl L, Kremmer E, Ellwart J, Sallusto F, Lipp M, Forster R. 2000. Follicular B helper 456 T cells express CXC chemokine receptor 5, localize to B cell follicles, and support 457 immunoglobulin production. J Exp Med 192:1545-52. 458 19. Schaerli P, Willimann K, Lang AB, Lipp M, Loetscher P, Moser B. 2000. CXC chemokine receptor 459 5 expression defines follicular homing T cells with B cell helper function. J Exp Med 192:1553- 460 62. 461 20. Hu Y, Smyth GK. 2009. ELDA: extreme limiting dilution analysis for comparing depleted and 462 enriched populations in stem cell and other assays. J Immunol Methods 347:70-8. 463 21. Christa E. Osuna RA, So-Yon Lim, Jessica L. Kublin, Rasmi Thomas, Yanqin Ren, Nathaniel D. 464 Bachtel, Margaret Ackerman, Jintanat Ananworanich, Dan Barouch, Nelson L. Michael, R. Brad 465 Jones, Douglas Nixon, James Whitney. 2018. CD32 does not mark the HIV-1/SIV latent 466 reservoir, abstr Conference on Retroviruses and Opportunistic Infections (CROI), Boston, 467 22. Pantaleo G, Graziosi C, Butini L, Pizzo PA, Schnittman SM, Kotler DP, Fauci AS. 1991. Lymphoid 468 organs function as major reservoirs for human immunodeficiency virus. Proc Natl Acad Sci U 469 S A 88:9838-42. 470 23. Pantaleo G, Graziosi C, Demarest JF, Butini L, Montroni M, Fox CH, Orenstein JM, Kotler DP, 471 Fauci AS. 1993. HIV infection is active and progressive in lymphoid tissue during the clinically 472 latent stage of disease. Nature 362:355-358. 473 24. Embretson J, Zupancic M, Ribas JL, Burke A, Racz P, Tenner-Racz K, Haase AT. 1993. Massive 474 covert infection of helper T lymphocytes and macrophages by HIV during the incubation 475 period of AIDS. Nature 362:359-62. 476 25. Schacker T, Little S, Connick E, Gebhard-Mitchell K, Zhang ZQ, Krieger J, Pryor J, Havlir D, Wong 477 JK, Richman D, Corey L, Haase AT. 2000. Rapid accumulation of human immunodeficiency 478 virus (HIV) in lymphatic tissue reservoirs during acute and early HIV infection: implications 479 for timing of antiretroviral therapy. J Infect Dis 181:354-7. 480 26. Haase AT, Henry K, Zupancic M, Sedgewick G, Faust RA, Melroe H, Cavert W, Gebhard K, 481 Staskus K, Zhang ZQ, Dailey PJ, Balfour HH, Jr., Erice A, Perelson AS. 1996. Quantitative image 482 analysis of HIV-1 infection in lymphoid tissue. Science 274:985-9. 483 27. Cavert W, Notermans DW, Staskus K, Wietgrefe SW, Zupancic M, Gebhard K, Henry K, Zhang 484 ZQ, Mills R, McDade H, Schuwirth CM, Goudsmit J, Danner SA, Haase AT. 1997. Kinetics of 485 response in lymphoid tissues to antiretroviral therapy of HIV-1 infection. Science 276:960-4.
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486 28. Reinhart TA, Rogan MJ, Huddleston D, Rausch DM, Eiden LE, Haase AT. 1997. Simian 487 immunodeficiency virus burden in tissues and cellular compartments during clinical latency 488 and AIDS. J Infect Dis 176:1198-208. 489 29. Estes JD, Kityo C, Ssali F, Swainson L, Makamdop KN, Del Prete GQ, Deeks SG, Luciw PA, 490 Chipman JG, Beilman GJ, Hoskuldsson T, Khoruts A, Anderson J, Deleage C, Jasurda J, Schmidt 491 TE, Hafertepe M, Callisto SP, Pearson H, Reimann T, Schuster J, Schoephoerster J, Southern P, 492 Perkey K, Shang L, Wietgrefe SW, Fletcher CV, Lifson JD, Douek DC, McCune JM, Haase AT, 493 Schacker TW. 2017. Defining total-body AIDS-virus burden with implications for curative 494 strategies. Nat Med 23:1271-1276. 495 30. Vandergeeten C, Fromentin R, DaFonseca S, Lawani MB, Sereti I, Lederman MM, Ramgopal M, 496 Routy JP, Sekaly RP, Chomont N. 2013. Interleukin-7 promotes HIV persistence during 497 antiretroviral therapy. Blood 121:4321-9. 498 31. Procopio FA, Fromentin R, Kulpa DA, Brehm JH, Bebin AG, Strain MC, Richman DD, O'Doherty 499 U, Palmer S, Hecht FM, Hoh R, Barnard RJ, Miller MD, Hazuda DJ, Deeks SG, Sekaly RP, Chomont 500 N. 2015. A Novel Assay to Measure the Magnitude of the Inducible Viral Reservoir in HIV- 501 infected Individuals. EBioMedicine 2:874-83. 502 32. Brennan TP, Woods JO, Sedaghat AR, Siliciano JD, Siliciano RF, Wilke CO. 2009. Analysis of 503 human immunodeficiency virus type 1 viremia and provirus in resting CD4+ T cells reveals a 504 novel source of residual viremia in patients on antiretroviral therapy. J Virol 83:8470-81. 505 33. Banga R, Procopio FA, Cavassini M, Perreau M. 2015. In Vitro Reactivation of Replication- 506 Competent and Infectious HIV-1 by Histone Deacetylase Inhibitors. J Virol 90:1858-71. 507
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517 FIGURE LEGENDS 518 519 Fig 1. CD32 expression on CD4 T cells in LN and blood of HIV-1 infected and uninfected
520 individuals. LN and PB mononuclear cells were isolated from the same HIV-uninfected (N=9),
521 HIV-1 infected ART treated (N=19) and HIV-1 infected viremic individuals (N=10). Cells were
522 stained with anti-CD3, anti-CD4, anti-CD45RA, anti-CD32 antibodies. (A) Representative flow
523 cytometry profiles of blood and LN memory (CD45RA-) CD4 T cell populations expressing CD32
524 in representative HIV-1 uninfected, ART treated and viremic subjects. (B) Cumulative data on the
525 frequencies of CD32+ memory CD4 T cells in blood and LN mononuclear cells of HIV-1
526 uninfected (green), ART treated (red) and viremic (blue) individuals. P values were obtained by
527 Mann-Whitney to compare the three groups and Wilcoxon signed-rank test to compare frequencies
528 between blood and LNs. **P<0.01, *** P<0.001, ****P<0.0001. Error bars denote mean ± S.E.M.
529
530 Fig 2. CD32+ CD4 T cells from LNs are enriched in PD-1+ and Tfh cells. (A) Representative
531 flow cytometry profiles of CD32 expression in LN memory CD4 T cell populations defined by
532 PD-1 and CXCR5 expression of HIV-1 uninfected, HIV-1 infected ART treated and viremic
533 individuals. (B) Cumulative data on the frequencies of CD32+ memory CD4 T cells within PD-1-
534 CXCR5-, PD-1-CXCR5+, PD-1+CXCR5- and Tfh (PD-1+ CXCR5+) cell populations isolated from
535 LNs of 9 HIV-1 uninfected, 19 ART treated and 10 viremic individuals. (C) Percentage of PD-1+
536 cells in gated CD32- and CD32+ memory (CD45RA-) CD4 T cell populations in the three groups.
537 (D) Distribution of Tfh within CD32- and CD32+ memory CD4 T cell populations. Triangles
538 represent HIV-1 uninfected, round circles represent ART treated and squares represent viremic
23 bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
539 individuals. *P<0.05, **P<0.01, ****P<0.0001 values were obtained by Wilcoxon signed-rank
540 test and error bars denote mean ± S.E.M.
541
542 Fig 3. Mass cytometry analysis of LN memory CD4 T cells defined by CD32 and/or PD-1
543 expression. Mass cytometry staining was performed in LN mononuclear cells isolated from 9 HIV-
544 1 uninfected, 16 HIV-1 infected ART treated and 9 viremic individuals. Cells were stained with a
545 panel of 30 cell surface markers (S1 Table). Heat maps of mean marker intensities in memory CD4
546 T cells defined on the basis of (A) CD32 and of (B) CD32 and PD-1 expression. (C) Venn diagram
547 summarizing common trends in marker expression between memory (CD45RA-) CD4 T cells
548 defined on the basis of PD-1 and CD32 expression. Black arrows indicate trends that are common
549 to all study groups (HIV uninfected, ART treated and viremic individuals). Green, red and blue
550 arrows indicate trends that are specific to each study group.
551
552 Fig 4. Levels of total HIV DNA and cell-associated HIV RNA in CD32+ and PD-1+ CD4 T cell
553 populations. (A) Quantification of total HIV DNA (copies per million cells) of sorted memory
554 (CD45RA-) CD4 T expressing or not CD32 and PD-1 from 7 aviremic ART treated (round circles)
555 and 7 viremic individuals (squares). (B) Frequencies of cells with inducible replication competent
556 HIV in sorted CD32-, PD-1- and total memory CD4 T cells estimated using conventional limiting
557 dilution methods by Extreme Limiting Dilution analysis
558 (http://bioinf.wehi.edu.au/software/elda/)(32, 33) in 3 aviremic ART treated individuals. (C)
559 Levels of cell-associated unspliced HIV RNA (copies/million cells) in sorted CD32-PD-1-,
560 CD32+PD-1-, CD32-PD-1+ and CD32+PD-1+ memory CD4 T cell populations isolated from 10
24 bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
561 aviremic ART treated and 6 viremic individuals. *P<0.05, **P<0.01 values were obtained by
562 Wilcoxon signed-rank test and error bars denote mean ± S.E.M.
563
564 Fig 5. Correlations between memory CD4 T cell populations defined by CD32 and/or PD-1
565 and parameters of disease activity. (A) Correlation between the percentage of CD32+ memory
566 CD4 T cells and CD4 T cell count, levels of viremia and years of suppressive ART. Correlation
567 between the percentage of CD32-PD-1-, CD32+PD-1-, CD32-PD-1+ and CD32+PD-1+ populations
568 and (B) levels of viremia (C) and years of suppressive ART. Spearman rank test was used for
569 correlations.
570
571 Fig 6. Correlation between memory CD4 T cell populations defined by PD-1 and parameters
572 of disease activity. Correlation between the percentage of PD-1+ memory CD4 T cells with CD4
573 T cell count, viral load and years of suppressive ART. Spearman rank test was used for correlations.
574
575 SUPPLEMENTAL MATERIAL
576 S1 Fig. Distribution of Tfh cells within CD32-, CD32+ and PD-1+ CD4 T cell populations
577 from lymph nodes. Triangles represent HIV-1 uninfected (N=9), round circles represent HIV-1
578 infected ART treated (N=19) and squares represent viremic individuals (N=10). ****P<0.0001
579 values were obtained by Wilcoxon signed-rank test and error bars denote mean ± S.E.M.
580
25 bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
581 S2 Fig. Frequencies of blood memory CD4 T cell populations on the basis of CD32 and PD-
582 1. (A) Representative flow cytometry profiles of blood memory (CD45RA-) CD4 T cell
583 populations expressing CD32 and/or PD-1 from representative HIV-1 uninfected, ART treated and
584 viremic individuals. (B) Cumulative data of the frequencies of CD32-PD-1-, CD32+PD-1-, CD32-
585 PD-1+, and CD32+PD-1+ CD4 T cell populations in blood mononuclear cells from HIV-1
586 uninfected (N=9), HIV-1 infected ART treated (N=16) and viremic individuals (N=10). **P<0.01
587 and *** P<0.001 values were obtained by Mann-Whitney test and error bars denote mean ± S.E.M
588
589 S3 Fig. Mass cytometry profiles of CD32 expressing memory CD4 T cells from lymph nodes.
590 Triangles represent HIV-1 uninfected (N=9), round circles represent HIV-1 infected ART treated
591 (N=16) and squares viremic individuals (N=9). *P<0.05, ****P<0.0001 values were obtained
592 using linear-effect mixed models with bonferroni correction for multiple testing and error bars
593 denote mean ± S.E.M.
594
595 S4 Fig. Mass cytometry analysis of LN memory CD4 T cells defined by CD32 and PD-1
596 expression. Triangles represent HIV-1 uninfected (N=9), round circles represent HIV-1 infected
597 ART treated (N=16) and squares viremics individuals (N=9). *P<0.05, **P<0.01, *** P<0.001,
598 ****P<0.0001 values were obtained using linear-effect mixed models with bonferroni correction
599 for multiple testing and error bars denote mean ± S.E.M.
600
601 S5 Fig. Gating strategies for cell sorting experiments. (A) gating strategy for CD32+ and CD32-
602 (left panel) and PD-1+ and PD-1- (right panel) memory (CD45RA-) CD4 T cell populations. (B)
26 bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
603 gating strategy for CD32-PD-1-, CD32+PD-1-, CD32-PD-1+, CD32+PD-1+ memory (CD45RA-)
604 CD4 T cell populations.
605
606 S6 Fig. Gating strategy for flow cytometry analysis. (A) In the gating of singlet memory CD4+
607 T cells, lymphocytes were first gated (SSC-A vs. FSC-A) followed by singlets (FSC-A vs. FSC-W
608 and SSC-A vs SSC-W). Lymphocytes were then gated for their uptake of the Live/Dead Aqua stain
609 to determine live versus dead cells and for expression of CD3. On the viable CD3+ cells, we then
610 excluded CD19 and CD8 expressing cells and gated on CD4+ cells. Low expression of CD45RA
611 was used to gate on memory CD4+ T cells followed by the measurement of CD32 and PD-1
612 expression. (B) Gated CD32-PD-1-, CD32+PD-1-, CD32-PD-1+, CD32+PD-1+ cells were
613 evaluated for the expression of CD19 and CD8 receptors.
614
615 S1 Table. Mass cytometry panel
27 bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 1
A HIV– HIV+ treated HIV+ viremic (H147) (MP068) (MP140) 105 105 105 0.3% 0.76% 0.5% 104 104 104
Blood 103 103 103
102 102 102
0 0 0 0102 103 104 105 0102 103 104 105 0102 103 104 105
105 105 105
4 1.99% 4 1.3% 4 4.46% Lymph 10 10 10 3 3 3 Node 10 10 10 102 102 102
0 0 0 CD4 0102 103 104 105 0102 103 104 105 0102 103 104 105 CD32 B ** 5 *** **** 4 ** HIV- HIV+ treated 3 HIV+ viremic CD4 T cells T CD4
+ 2 Percentage of Percentage
CD32 1
0 Blood LN bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 2
A Tfh CXCR5– PD-1– CXCR5+ PD-1– CXCR5– PD-1+ CXCR5+ PD-1+
105 105 105 105 1.85% 2.64% 4.6% 5.12% 4 4 4 4 HIV- LN 10 10 10 10 103 103 103 103 (008) 102 102 102 102 0 0 0 0
0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105
105 105 105 105 0.53% 0.49% 2.2% 2.99% 104 104 104 104 HIV+ Treated LN 103 103 103 103 (MP137) 102 102 102 102 0 0 0 0
0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105
105 105 105 105 + 0.43% 1.2% 3.4% 9.8% HIV Viremic LN 104 104 104 104
(MP124) 103 103 103 103 102 102 102 102 0 0 0 0 CD4 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 CD32
B HIV– LN HIV+ Treated LN HIV+ Viremic LN ** + 15 + 15 + 15 ** ** ** **** ** * **** ** 10 ** 10 **** 10 ** ** ****
5 5 5 CD4 T cells T CD4 cells T CD4 cells T CD4 Percentage of CD32 of Percentage 0 CD32 of Percentage 0 CD32 of Percentage 0 PD-1 – – ++ PD-1 – – ++ PD-1 – – ++ CXCR5 – ++– CXCR5 – ++– CXCR5 – ++– C D 80 ** 40
+ **** ** **** – 60 30 HIV LN **** HIV+ treated LN + 40 20 HIV viremic LN
CD4 T cells T CD4 ** 20 10 Percentage of PD-1 of Percentage
0 Tfh of Percentage cells 0 CD32 – ++– + CD32 –– + – ++– bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not Figure 3 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A
CXCR4 CCR7 ICOS CD27 CD127 HLADR CXCR5 CXCR3 CD57 CD40L CD38 PD1 CCR5 CCR6 CCR4 CD25 1.5 1 HIV− LN + + CD32 0.5 HIV Treated LN HIV− Viremic LN 0
− CD32 −0.5 p < 0.05 −1 p > 0.05 −1.5
B ICOS HLA-DR CXCR4 CD40L CD27 CD57 CD38 CXCR5 CXCR3 CCR5 CD127 CD25 CCR6 CCR7 CCR4 a. a. CD32-PD1+ vs. CD32+PD1+ b. b.CD32+PD1- vs. CD32+PD1+ c. c. CD32+PD1- vs. CD32-PD1+ d. d.CD32+PD1+ vs. CD32-PD1- e. e.CD32-PD1- vs. CD32-PD1+ f. f. CD32-PD1- vs. CD32+PD1- 2 CD32+PD1+
1 HIV− LN CD32-PD1+ HIV+ Treated LN − 0 HIV Viremic LN CD32+PD1- p < 0.05 −1 p > 0.05 CD32-PD1- −2
C – + PD-1 CD32 PD-1+ CD32– – – PD-1 CD32 PD-1+ CD32+ CXCR5 CD38 CXCR5 CD25 CD25 CXCR3 CXCR3 HLA-DR CD38 CCR7 CD40L ICOS ICOS CD127 CD127 CD27 CCR5 CCR5 CXCR4 CXCR4 CD57 CD57
CD40L CD40L Up-regulated (in all groups) CD27 Down-regulated (in all groups) HIV– LN CD25 CCR6 HIV+ Treated LN HIV+ Viremic LN bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 4 A B HIV+ Treated LN HIV+ Viremic LN 40 8000 106 * * 5 30 6000 * 10 ns 20 4000 104
10 2000 103 inducible replication
2 Frequency of cells with 0 HIV DNA (copies/million HIV DNA cells ) (copies/million HIV DNA cells ) 0 10 competent virus (RUPM) – + – + – + – + – –
PD-1 PD-1 CD32 PD-1 C CD32 CD32 PD-1 CD32 CD32 PD-1 ** * Tot memory 100000 ** 107 * * * * 6 * 80000 10 * 105 60000 HIV+ treated LN ** 4 10 HIV+ viremic LN 40000 103 20000 102 (copies/million cells ) (copies/million cells ) Cell associated HIV RNA Cell associated HIV RNA 0 101 CD32 –– ++ CD32 –– ++ PD-1 – + +– PD-1 – + +– bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 5
A r = -0.58 r = 0.68 r = -0.79 ) 3 1500 P = 0.0008 106 P < 0.0001 20 P < 0.0001
5 10 15 1000 HIV+ Treated LN 104 10 HIV+ Viremic LN 103 500 5 102
0 101 0 Viral load (copies/ml) Viral
CD4 count (cells/mm 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 Years of suppressive ART of suppressive Years % of CD32+ CD4 T cells % of CD32+ CD4 T cells % of CD32+ CD4 T cells B r = -0.46 r = 0.56 r = 0.46 r = 0.65 P = 0.01 P = 0.001 P = 0.03 P = 0.0001 106 106 106 106
105 105 105 105
104 104 104 104
103 103 103 103
102 102 102 102
Viral load (copies/ml) Viral 101 load (copies/ml) Viral 101 load (copies/ml) Viral 101 load (copies/ml) Viral 101 50 60 70 80 90 100 0 1 2 3 0 10 20 30 40 50 0 1 2 3 4 % of CD32–PD-1– CD4 T cells % of CD32+PD-1– CD4 T cells % of CD32–PD-1+ CD4 T cells % of CD32+PD-1+ CD4 T cells C
20 r = 0.6 20 r = -0.67 20 r = -0.56 20 r = -0.8 P = 0.0004 P < 0.0001 P = 0.001 P < 0.0001 15 15 15 15
10 10 10 10
5 5 5 5
0 0 0 0 50 60 70 80 90 100 0 1 2 3 0 10 20 30 40 50 0 1 2 3 4 Years of suppressive ART of suppressive Years ART of suppressive Years ART of suppressive Years ART of suppressive Years % of CD32–PD-1– CD4 T cells % of CD32+PD-1– CD4 T cells % of CD32–PD-1+ CD4 T cells % of CD32+PD-1+ CD4 T cells bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 6
) r = -0.51 6 r = 0.43 3 1500 10 P = 0.004 P = 0.01 105 1000 104 103 500 102
Viral load (copies/ml) load Viral 1 CD4 count (cells/mm CD4 count 0 10 0 10 20 30 40 50 0 10 20 30 40 50 Percentage of PD-1+ CD4 T cells Percentage of PD-1+ CD4 T cells
20 r = -0.58 HIV+ Treated LN P = 0.0009 HIV+ Viremic LN 15
10
5
0 Years of suppressive ART suppressive of Years 0 10 20 30 40 50 Percentage of PD-1+ CD4 T cells bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
S1 Figure
**** 60 **** HIV– LN HIV+ Treated LN 40 **** HIV+ Viremic LN
20
Percentage of Tfh of Percentage cells 0 - + +
CD32 CD32 PD-1 bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
S2 Figure A Gated on CD4+CD45RA–
+ HIV– HIV treated HIV+ viremic (008) (MP137) (MP106)
105 3.18% 0.02%105 6.89% 0.06% 105 20.3% 0.2%
104 104 104
103 103 103
102 102 102
0 96.5% 0.3% 0 92.6% 0.44% 0 79.1% 0.43% PD-1 0102 103 104 105 0102 103 104 105 0102 103 104 105 CD32 B
100 *** 1.5 80
D4 T cells T D4 – C
CD4 T cells 1.0
- HIV - 60 HIV+ treated PD-1 PD-1 - 40 + + 0.5 HIV viremic 20 % of CD32 0 CD32 % of 0.0
*** 25 ** 1.0 20 0.8 CD4 T cells T CD4 CD4 T cells T CD4 + + 15 0.6 PD-1 PD-1 - 10 + 0.4 5 0.2 % of CD32 % of 0 CD32 % of 0.0 bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
S3 Figure
CXCR5 CXCR3 CCR6 CD38
80 **** 80 **** 100 **** 100 ****
80 80 60 60 60 60 40 40 CD4 T cells T CD4 cells T CD4 CD4 T cells T CD4 CD4 T cells T CD4
+ + 40 40 + + Percentage of Percentage Percentage of Percentage of Percentage 20 20 of Percentage 20 20 CD38 CCR6 CXCR5 CXCR3 0 0 0 0 CD32– CD32+ CD32– CD32+ CD32– CD32+ CD32– CD32+ ICOS CCR4 CD27 CD25
80 **** 80 **** 100 80 ****
80 60 60 60 60 40 40 40 CD4 T cells T CD4 CD4 T cells T CD4 CD4 T cells T CD4 CD4 T cells T CD4 40 + + + + Percentage of Percentage Percentage of Percentage Percentage of Percentage Percentage of Percentage 20 20 20 20 ICOS CD25 CD27 CCR4 0 – + 0 0 0 CD32 CD32 CD32– CD32+ CD32– CD32+ CD32– CD32+
CCR7 CD40L CCR5 HLA-DR **** **** 100 100 **** 50 80
80 80 40 60 60 60 30
CD4 T cells T CD4 40 CD4 T cells T CD4 CD4 T cells T CD4 CD4 T cells T CD4 +
+ 20 40 + + 40 Percentage of Percentage Percentage of Percentage Percentage of Percentage Percentage of Percentage 20 20 20 10 CCR5 CCR7 CD40L HLA-DR 0 0 0 0 CD32– CD32+ CD32– CD32+ CD32– CD32+ CD32– CD32+ CXCR4 CD57 CD127 PD-1 **** * 50 50 100 80 ****
40 40 80 60 30 30 60 40 CD4 T cells T CD4 CD4 T cells T CD4 CD4 T cells T CD4 CD4 T cells T CD4 + 20 20 + 40 + + Percentage of Percentage Percentage of Percentage Percentage of Percentage Percentage of Percentage 20 10 10 20 PD-1 CD57 CD127 CXCR4 0 0 0 0 – CD32– CD32+ CD32– CD32+ CD32– CD32+ CD32 CD32+
HIV– LN HIV+ treated LN HIV+ viremic LN bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
S4 Figure CXCR5 CXCR3 CCR6 CD38 **** **** **** **** **** **** *** 100 **** 100 **** 80 **** 100 ** ***** **** **** **** ** ******** 80 80 80 *** 60 60 60 60 40 CD4 T cells T CD4 cells T CD4 CD4 T cells T CD4 CD4 T cells T CD4
+ 40 + 40 40 + + Percentage of Percentage Percentage of Percentage Percentage of Percentage 20 of Percentage 20 20 20 CD38 CCR6 CXCR5 CXCR3 0 0 0 0
ICOS CCR4 CD27 CD25 ******** **** * *** 100 **** 100 100 80 *** ** **** 80 80 80 ** 60 60 60 60 40 CD4 T cells T CD4 CD4 T cells T CD4 cells T CD4 CD4 T cells T CD4 40
40 + 40 + + + Percentage of Percentage Percentage of Percentage Percentage of Percentage Percentage of Percentage 20 20 20 20 ICOS CD27 CD25 CCR4 0 0 0 0
CCR7 CD40L CCR5 HLA-DR **** **** **** ******** **** 100 60 80 **** 80 *** **** **** **** **** **** 80 **** **** **** **** 60 *** 60 **** 40 60
40 TCD4 cells 40 CD4 T cells T CD4 CD4 TCD4 cells CD4 T cells T CD4 + +
40 + + 20 Percentage of Percentage Percentage of Percentage Percentage of Percentage Percentage of Percentage 20 20 20 CCR5 CCR7 CD40L HLA-DR 0 0 0 0
CXCR4 CD57 CD127 **** **** – ******** **** HIV LN 80 80 **** 100 **** + **** **** ** HIV treated LN * 80 HIV+ viremic LN 60 60 60 40 – – CD4 T cells T CD4 40 CD4 T cells T CD4 PD-1 CD32 CD4 T cells T CD4 + + – + + 40 PD-1 CD32 + – Percentage of Percentage Percentage of Percentage Percentage of Percentage 20 20 20 PD-1 CD32
CD57 + + CD127
CXCR4 PD-1 CD32 0 0 0 bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
S5 Figure
A Gated on CD4+CD45RA–
105 CD32– CD32+ 105 PD-1+ 104 104
103 103
0 0 PD-1– PD-1 PD-1 0 103 104 105 0 103 104 105 CD32 CD32 B Gated on CD4+CD45RA–
105 CD32–PD-1+ CD32+PD-1+
104
103
0
CD32–PD-1– CD32+PD-1– PD-1 0 103 104 105 CD32 bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
S6 Figure A!
250K 250K 250K
200K 200K 200K
150K 150K 79.4 150K 96.4
98.9 100K 100K 100K SSC-A ! FSC-W ! SSC-W ! 50K 50K 50K
0 0 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K
FSC-A! FSC-A! SSC-A!
5 10 105 105
61.4 4 10 104 104
3 3 10 10 103
2 10 102 102 0 69.5 0 0
82.7 CD4(AF700) ! CD19/CD8(PB) ! Live/Dead(Aqua) !
2 3 4 5 0 10 10 10 10 0 102 103 104 105 0 102 103 104 105 CD3 (APC-Cy7)! CD4 (AF700)! CD45RA (ECD)!
17.1 0.347 105
104
103
0 PD-1(Pe-Cy7) !
82.1 0.515
0 102 103 104 105 CD32 (APC)! bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
B! CD32-PD-1- CD32+PD-1- CD32-PD-1+ CD32+PD-1+
0 0 0 0 0 0 0 0 105 105 105 105
104 104 104 104
103 103 103 103
102 102 102 102 0 0 0 0
0 100 0 100 0 100 0 100
CD19/CD8(PB) ! 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD4 (AF700)! bioRxiv preprint doi: https://doi.org/10.1101/329938; this version posted May 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
S1 Table
Target Metal Company Clone CD4 115In Biolegend RPA-T4 CCR6 141Pr Fluidigm/DVS G034E3 CD19 142Nd Fluidigm/DVS HIB19 ICOS 143Nd Biolegend C398.4A CD8 145Nd Biolegend RPA-T8 IgD 146Nd Fluidigm/DVS IA6-2 CD7 147Sm Fluidigm/DVS CD7-6B7 CD57 148Nd BD G10F5 CCR4 149Sm Fluidigm/DVS 205410 CXCR5 153Eu Fluidigm/DVS RF8B2 CD21 152Sm Fluidigm/DVS BL13 CXCR3 154Sm Biolegend G025H7 CD27 155Gd Fluidigm/DVS L128 CD11c 156Gd Biolegend 3.9 CCR7 159Tb Fluidigm/DVS G043H7 CD25 158Gd Biolegend M-A251 CD14 160Gd Fluidigm/DVS M5E2 CD1C 161Dy Biolegend L161 CD32-APC 162Dy Fluidigm/DVS FUN2 CD20 166Er Fluidigm/DVS 2H7 CD38 167Er Fluidigm/DVS HIT2 CD45RA 169Tm Fluidigm/DVS HI100 CD40L 168Er Fluidigm/DVS CD40L CD3 170Er Fluidigm/DVS UCHT1 CCR5 171Yb Fluidigm/DVS NP-6G4 HLA-DR 173Yb Fluidigm/DVS L243 PD-1 174Yb Fluidigm/DVS EH12.2H7 CXCR4 175Lu Fluidigm/DVS 12G5 CD127 176Yb Fluidigm/DVS A019D5 CD16 209BI Fluidigm/DVS 3G8 Live/Dead 195Pt Fluidigm/DVS Cell-ID