PD-1 and its ligand PD-L1 are progressively up-regulated on CD4 and CD8 T-cells. in HIV-2 infection irrespective of the presence of viremia

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1 Tendeiro et al_ Supplemental Digital ontent_page of PD and its ligand PDL are progressively upregulated on D4 and D8 Tcells in infection irrespective of the presence of viremia Supplemental Digital ontent Rita TENDEIRO *, Russell B. FOXALL *, António P. BAPTISTA, Francisco PINTO, Rui S. SOARES, Rita AVALEIRO, Emília VALADAS, Perpétua GOMES,4,5, Rui M. M. VITORINO,6, Ana E. SOUSA * The authors contributed equally to this work. Unidade de Imunologia línica, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. línica de Doenças Infecciosas, Hospital de Santa Maria, Lisboa, Portugal. Laboratório de Biologia Molecular, Serviço de Medicina Transfusional, entro Hospitalar Lisboa Ocidental, Hospital Egas Moniz, Lisboa, Portugal. 4 entro de Malária e Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal. 5 entro de Investigação Interdisciplinar Egas Moniz (iiem), Instituto Superior de iências da Saúde Sul, aparica, Portugal. 6 línica Universitária de Medicina, entro Hospitalar Lisboa Norte, Hospital Universitário de Santa Maria, Lisboa, Portugal. orresponding Author: Ana E. Sousa, MD PhD Unidade de Imunologia línica, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 6498 Lisboa, Portugal. Phone: , Fax: asousa@fm.ul.pt

2 Tendeiro et al_ Supplemental Digital ontent_page of Supplemental Table. ohort clinical and epidemiological data. a atives HIV Numbers (male/female) 6 (6/) (7/5) 8 (9/9) Age (years) 44 ± (757) 4 ± (6) 49 ± (978) Ethnicity (aucasian/other) 5/ /7 4/4 % D cells 6.6 ±.9 (4976.) 67.4 ±. ( ) 6.8 ±. ( ) D cells/µl 86 ± 9 (69599) 6 ± 7 (5897) 99 ± (469) % D4 Tcells 5.4 ±.5 (5.667.) 4.6 ±.9 (.77.)***.6 ±. (6.86)*** D4 Tcells/µl 96 ± 59 (58) 569 ± 5 (8848)* 659 ± 8 (55)* % HLADR in D4 4. ±.4 (.97.6) 7. ±. (.754.5)***.4 ±.7 (.86.)** % D8 Tcells 4. ±.8 (.75.) 46.7 ±.8 (.464.7)*** 8. ±. (65.7)***# D8 Tcells/µl 5 ± 5 (8) 7 ± (854)*** 86 ± 75 (87)**# D8 MFI in D8 7 ± 6 (8859) 58 ± 848 (7655)*** 854 ± 57 (64659)* # (RNA cp/ml) NA ± 4956 (74.5x 6 ) b 66 ±,6 (89.6x 4 ) c a Data are Mean±SEM with limits in brackets, unless indicated otherwise. NA, Not Applicable. Data referring to the early stage cohort are not included in this Table (see Supplemental Table ). Statistical differences between a given HIV cohort and the seronegative controls: *, p < 5; **, p < ; ***, p <. Statistical differences between HIV and individuals: #, p < 5. b HIV viremia was < 4 RNA copies/ml (cutoff) in of the subjects studied. c viremia was < RNA copies/ml (cutoff) in of the 8 subjects studied.

3 Tendeiro et al_ Supplemental Digital ontent_page of Supplemental Table. orrelations between PD or PDL MFI on D4 and D8 T cells and additional surrogate markers of HIV disease progression. a PD MFI atives HIV D4 D8 D4 D8 D4 D8 HLADR MFI in D4.6;.7.4;..7;.6;..67;<.45; %HLADR in D8.6;.55.7;.5.47;.4;4.6;4.7;5 HLADR MFI in D8.;.9.;.4.5;..58;5.6;8.47; %D8 in D8.;.68.;.67.55;9.5;.46;.44; βmicroglobulin (mg/l) b ND ND.8;.8.;.7.57;75.5;. PDL MFI atives HIV D4 D8 D4 D8 D4 D8 HLADR MFI in D4.7;.5.4;..8;..8;..5;4.;9 %HLADR in D8.6;.54 ;.97.56;.5;.5;5.9;4 HLADR MFI in D8.;.68 ;.98.4;6.44;4.49;8.4;7 %D8 in D8.;.5.7;.6.8;..;.7.5;4.;8 βmicroglobulin (mg/l) b ND ND.7;.5 ;.98.66;.55; a Relationships were tested for significance with Spearman correlations: r;p values are shown. Results with p values <5 are highlighted in bold. ND: Not Determined. b βmicroglobulin serum levels were assessed for (.±.4 mg/l) and 4 HIV (.±.4 mg/l) infected individuals, with no statistically significant differences having been observed between cohorts.

4 Tendeiro et al_ Supplemental Digital ontent_page 4 of Supplemental Table. linical and epidemiological data of the early stage cohorts. a atives HIV a a Numbers (male/female) 8 (/5) (8/4) 7 (/6) Age (years) 4 ± 5 (657) 4 ± (56) 5 ± 5 (66) Ethnicity (aucasian/other) 8/ 9/ 5/ % D cells 67. ±.8 (5.888.) 69.7 ±. ( ) 7. ±. ( ) D cells/µl 75 ± 7 (975) 7 ± (756) 547 ± 97 (657) % D4 Tcells 6. ±.6 (6.5.9) 6.9 ±.6 (9.45.)*.6 ±. ( ) D4 Tcells/µl 95 ± 8 (585) 66 ± 74 (46) 697 ± 4 (5464) % HLADR in D4 5.8 ±.9 (.8.) 6.7 ±. (.5.7) 5.9 ±.4 (..6) % D8 Tcells.4 ± 5.4 (.6.) 4. ±.9 (6.67)* 4.4 ±.9 (8.47.7) D8 Tcells/µl 88 ± 89 (75) 54 ± 4 (49887) 84 ± 76 (56) D8 MFI in D8 447 ± 98 (9499) 4895 ± 66 ( )** 98 ± (454665) (RNA cp/ml) NA 5,949 ± 845 (957,) NA b a Data are Mean±SEM with limits in brackets, unless indicated otherwise. NA, Not Applicable. Statistical differences between a given HIV cohort and the seronegative controls: *, p < 5; **, p <. b viremia was < RNA copies/ml (cutoff) in all of the subjects studied.

5 PD MFI in D8 PD MFI in D4 PD MFI in D8 subsets PD MFI in D4 subsets D8 % of Max % of Max PD MFI in D8 Tendeiro et al_ Supplemental Digital ontent_page 5 of Supplemental Figure. PD expression according to celldifferentiation and viremia in individuals. A Gated on D cells B r=.89 p= HIV r=.47 p=6 r=.657 p= 5 5 D4 5 PD PD MFI in D < < 7 9 < < < 8 9 < < < < D < < 5 5 HIV D45RO D7 D45RO D7 D45RO D7 D45RO D7 HIV D45RO D7 D45RO D7 D45RO D7 E Avir Vir Avir Vir (A) Gating strategy used to assess PD (clone: MIH4) expression by flow cytometry: cells were first gated according to D8 or D4 expression (within a D gate) and PD expression was then assessed using as reference a fluorescence minus one control (grey histogram). (B) Similarly to seronegative controls, individuals exhibited a significant correlation between PD expression on D8 and on D4 Tcell subsets. PD expression was assessed within naïve and memory D8 () and D4 (D) Tcell subsets defined according to D45RO and D7 expression. Bars represent mean±sem and statistically significant differences are shown. (E) PD expression in individuals stratified according to levels of circulating virus: undetectable (aviremic, avir) and viremic (vir). Each dot represents one individual.

6 Tendeiro et al_ Supplemental Digital ontent_page 6 of Supplemental Figure. anonical correlation analysis. As none of the MLR models fully explained the variability in PD expression seen among the different Tcell subsets (r <.6 for all models), a canonical correlation analysis was performed in order to clarify the determinants of PD expression within Tcells. This technique looks for correlations between linear combinations of a set of predictive variables and of outcome variables, commonly denominated variates or canonical variables. In our analysis, the predictive variate (variate ) included D4 Tcell counts, viral load and Tcell activation (HLADR expression on D4 Tcells and HLADR and D8 expression on D8 Tcells, measured as MFI), and the outcome variate (variate ) PD expression (MFI) on D4 and D8 Tcells in all the individuals studied. The two most significant correlations found are shown. A r=.77 p=x HIV B... PD MFI (in D4) PD MFI (in D8).5 HIV. The first (A) shows a strong association between a predictive variate dominated by Tcell.5.5 activation parameters and a outcome variate with high values for PD expression on.5 PD MFI (in D4) PD MFI (in D8) r=.64 p=x 6 both total D4 and D8 Tcells, which implicates immune activation as the major determinant of PD expression on both Tcell subsets r=.779 p=5x HIV..4 ounts iremia D4) D8) D8).

7 Variable we Variable.. B...5. Tendeiro et al_ Supplemental Digital ontent_page 7 of PD MFI (in D4).5.5 PD MFI (in D8) r=.64 p=x 6 HIV..5 PD MFI (in D4) PD MFI (in D8).6 The second (B) represents an association p=5x. between low HLADR expression on HIV D4 T.4.5. cells and high viremia with low PD expression on D4 Tcells and high PD. expression on D8 Tcells, thus reinforcing the role of immune activation in driving PD.4. PD (D4 D45RO T cells) PDMFI(D8 D45RO D7 cells) PDMFI(D8 D45RO D7 cells) PDMFI(D8 D45RO D7 cells) r=.779 overexpression but also indicating a contribution of viremia in the upregulation of PD on D8 Tcells. D r=.569 olinearity analysis failed to reveal any significant association between PD expression..5 p=.6x on the different.5 Tcell subpopulations, which suggests that PD expression is regulated.5 in a subsetindependent manner. To address the regulation of PD expression within the HIV..5 different Tcell subpopulations, we performed an additional canonical correlation analysis in which disease progression markers (variate ) were correlated to PD expression on memoryeffector D4 and D8 Tcell subsets (variate among memory PD MFI (D4D45RO T cells) PDMFI(D8D45ROD7cells) PDMFI(D8D45ROD7cells) PDMFI(D8D45ROD7cells) D4 Tcells, D45RO cells were considered as a whole as no difference in PD expression was observed between D7 and D7 neg counterparts). The two most significant correlations found are shown.

8 Variable weight Variate Variable weight Variable weight Variate Variable Variab weig Variable Varia wei Variate B Tendeiro et al_ Supplemental Digital ontent_page 8 of.5 PD MFI (in D4) PD MFI (in D4) PD MFI (in D8) PD (D4 D45RO T cells) PDMFI(D8 D45RO D7 cells) PDMFI(D8 D45RO D7 cells) PDMFI(D8 D45RO D7 D4 D45RO. and D8 D45RO.5 D7 Tcells...5 cells) r=.64 p=x 6 r=.779 p=5x Variate HIV HIV r=.779 D.6 p=5x. r=.569 HIV.4 p=.6x HIV The first () demonstrates a relationship between a predictive variate combining mostly.5.5. Tcell.4 activation parameters and a outcome variate combining PD expression within. D..5 PD MFI (D4D45RO T cells) PD MFI (in D8) PD MFI (in D4) PD MFI (in D8) PD (D4 D45RO T cells) PDMFI(D8 D45RO D7 cells) PDMFI(D8 D45RO D7 cells) PDMFI(D8 D45RO D7 PDMFI(D8D45ROD7cells) PDMFI(D8D45ROD7cells) PDMFI(D8D45ROD7cells).5.5 cells) r=.569 p=.6x Variate HIV..5 PD (D4D45RO T PDMFI(D8D45ROD7cells) PDMFI(D8D45ROD7cells) PDMFI(D8D45ROD7cells) The second (D) represents direct associations between D4 T cell counts, viremia and PD expression on D8 D45RO D7 Tcells and D4 T cell activation (HLADR expression) and PD expression on memory D4 Tcells. Overall, this analysis supports a major role for Tcell activation in PD upregulation on both D4 and D8 memory Tcells and a contribution of D4 Tcell depletion and viremia to PD overexpression on D8 D45RO D7 Tcells.

9 PD MFI in D8 subsets PD MFI in D4 subsets PDL MFI in D8 PDL MFI in D4 PDL MFI in D8 Tendeiro et al_ Supplemental Digital ontent_page 9 of Supplemental Figure. PDL expression on Tcells. A 5 5 B 5 r=.559 p=.76 HIV r=.86 p< r=.8 p< HIV HIV 5 5 PDL MFI in D < < 7 9 < < < 8 9 < < < < D < < 5 5 HIV D45RO D7 D45RO D7 D45RO D7 D45RO D7 HIV D45RO D7 D45RO D7 D45RO D7 (A) PDL MFI (clone MIH) within total D8 and D4 Tcells in seronegative, HIV and cohorts. Bars represent mean and each dot represents one individual. (B) Direct correlations were found in both infected cohorts between PDL expression levels within D4 and D8 Tcells. PDL expression was further addressed on naïve and memory D8 () and D4 (D) Tcell subsets in a group consisting of 4 seronegative, 5 HIV and individuals. Naïve and memory Tcell subsets were defined according to D45RO and D7 expression and PDL Mean Fluorescence Intensity (MFI) was then assessed within each cell population. Bars represent mean±sem.

10 TIM MFI in D8 subsets Tendeiro et al_ Supplemental Digital ontent_page of Supplemental Figure 4. TIM expression on D8 Tcell subsets D45RA D7 D45RA D7 D45RA D7 D45RA D7 HIV Graph shows TIM MFI (clone: 448) within each D8 Tcell subpopulation in all cohorts. Bars represent mean±sem.

11 TIM MFI PD MFI PDL MFI Tendeiro et al_ Supplemental Digital ontent_page of Supplemental Figure 5. PD, PDL and TIM expression on regulatory, memory Foxp neg, and naïve D4 Tcells. D45RA FoxP D4 T cells D45RA FoxP D4 T cells D45RA D4 T cells HIV HIV HIV TIM (left), along with PD (middle) and PDL (right) MFI was assessed within D45RA FoxP and D45RA FoxP populations and FoxP D4 Tcells in seronegative, HIV and individuals. Bars represent mean±sem. The results obtained with naïve and memory FoxP subsets were broadly similar to those observed within total D4 Tcells. Regarding the Treg population, both and HIV cohorts featured significantly higher levels of PD expression than the seronegative individuals, but TIM levels were similar in all cohorts. Also, a preliminary analysis revealed a strong correlation between PDL MFI on D4 Tcells and the frequency of FoxP expressing D4 Tcells in the (p=7, r=.88, n=8) but not in the HIV cohort (p=.88, r=.4, n=6).

Major Depletion of Plasmacytoid Dendritic Cells in HIV-2 Infection, an Attenuated Form of HIV Disease

Major Depletion of Plasmacytoid Dendritic Cells in HIV-2 Infection, an Attenuated Form of HIV Disease Rita Cavaleiro 1, António P. Baptista 1, Rui S. Soares 1, Rita Tendeiro 1, Russell B. Foxall 1, Perpétua