0 20 40 60 80

Figure 1

Percentage of positive cells analyzed by flow citometry in fresh PBMCs. These results show a significantly (p<0,001) inverse expression of CD4+ CD8+ cells in HIV+ patients in comparison to the healty donors phenotype.

0 20 40 60 80

HIV- HIV+

% of pos it iv e c el ls

CD4+

CD8+

p<0,001

Figure 2

Correlation between the number of CD4+ cells and the Viral Load in HIV+ patients. An increase of CD4+ cells numbers is correlated to a significant decrease of VL (Spearman’s rho = -711; p< 0,014).

0 1 2 3 4 5 6

0 500 1000

CD4+ cell count/mm ³

V ir al L o ad ( L og c opi es /m L ) . _p<0,014

Humoral Immune response 12%

Inflammatory

response 32% Cytokine chemokine

and receptors 41%

Other genes involved in cytokine- cytokine receptor interaction 15%

38%

18% 20%

24%

29%

14%

16%

40%

Figure 3

a) Distribution of functional gene grouping in “Autoimmune and Inflammatory response” microarray systems.

b) Pie chart summary of Tα1 regulated genes in super array experiments in PBMC from HIV+ patients.

c) Pie chart summary of Tα1 regulated genes in super array experiments in PBMC from healthy donors.

a)

b)

c)

0 20 40 60 80

up down

Percentage of m odulated genes

HIV+ HIV-

Figure 4

Number of positively and negatively modulated genes by Thymosin alpha 1 in PBMCs from HIV+ patients and healthy donors. 94 genes result regulated in HIV + patients, 64% are up regulated and 36% are down regulated; in HIV- donors the total of regulated genes are 74 whose 62% positively modulated and 38% negatively modulated.

n. 94 n. 74

Figure 5

Cytokine, Chemokine and receptors.

Expression of positively modulated genes (red) and negatively modulated genes (green) after treatment with Thymosin alpha 1 in PBMCs from HIV+ patients (a) and HIV- donors (b).

a) HIV+

Fold Change (T α 1 v s CTR)

b) HIV-

CCL2 CCL5 CCL7 CCL17 CCL19 CCL4L1 TNFSF11 TNFSF13B TRAP1 VEGFB BLR1 IL11RA IL4R IL7R TTN CCL11 CCL1 CCL3 CXCL2 CXCL12 CXCL10 CXCL16 IL8 CKLF CSF3R GHR IL3RA IL2RA

-1 1 3 5 7

CCL2 CCL3 CCL3L1 CCL4L1 CCL5 CCL18 CCL22 IL8 TRAP1 VEGFB CCR1 CCR7 CCRL2 IL4R IL6R IL10RA IL10RB CCL1 CCL11 CMTM1 CXCL1 IFNA1 IFNA 2 IL5RA LEFTY1 LEFTY2 OSMR IL2RG CCR2 CXCR6

-1 1 3

Figure 6

Cytokine-cytokine receptors interaction.

Expression of positively modulated genes (red) and negatively modulated genes (green) after treatment with Thymosin alpha 1 in PBMCs from HIV+ patients (a) and HIV- donors (b).

Fold Change (T α 1 vs CTR)

a) HIV+

b) HIV-

CCR1 CSF1R CSF2 CSF3 R CXCL16 ECGF1 GDF2 GRN IFNA8 LTB MIF TNFRSF1A TNFRSF1B TNFRSF8 CD86 SPN ACVR1 SLCO1A2 TGFB2 TGFBR2 TNFRSF9 TGFB3 XCR1 B7H3 CD28

-1 1 3

CSF1R GRN LTA OSM TNFRSF1A TNFRSF1B CD86 SIGIRR CLC IFNA1 IL1RN IL5 SDCBP SPRED1

-1 1 3 5

Figure 7

Humoral immune response.

Expression of positively modulated genes (red) and negatively modulated genes (green) after treatment with Thymosin alpha 1 in PBMCs from HIV+ patients (a) and HIV- donors (b).

Fold Change (T α 1 vs CTR)

a) HIV+

b) HIV-

BATF C3 CCL2 CCL3 CCL18 CCL22 CD27 CD53 CD74 CD97 CD180 CCR1 CCR7 CCRL2 CSF1R CYBB ITGB2 MIF SERPING1 BLNK MAPK14 SLA2

-1 1 3 5

CCL2 CCL7 CD86 CSF1R CYBB IL7R ITGB2 LY96 POU2F2 SERPING1 YY1 BLNK CCL3 CLC CD22 CD1C

-1 1 3 5 7

Figure 8

Inflammatory response.

Expression of positively modulated genes (red) and negatively modulated genes (green) after treatment with Thymosin alpha 1 in PBMCs from HIV+ patients.

Fold Change (T α 1 v s CTR)

HIV+

AIF1 ALOX5 C3 CCL2 CCL3L1 CCL4L1 CCL5 CCL3 CCL18 CCL22 CCR7 CD14 CD74 CEBPB CYBB CXCL16 FOS HDAC4 IL8 IL10RB IRF7 ITGAL ITGB2 LTA4H MYD88 PLA2G7 PRDX5 PREX1 S100A8 S100A12 STAB1 TLR2 CEBPB FN1 LBP BLNK CCL1 CCL11 IFNA 2 ICEBERG IL8RB NR3C1 OLR1 PTPRA SELE TACR1 TLR8

-1 1 3

Figure 9

Inflammatory response.

Expression of positively modulated genes (red) and negatively modulated genes (green) after treatment with Thymosin alpha 1 in PBMCs from healthy donors.

Fold Change (T α 1 vs CTR)

HIV-

AIF1 CD14 CCL2 CCL5 CCL7 CCL17 CCL4L1 CYBB FOS IRF7 ITGAL ITGB2 LY96 NFE2L1 NFATC3 PARP4 PREX1 S100A12 S100A8 STAB1 SIGIRR BLNK CCL11 CCL1 CCL3 CKLF CXCL2 CXCL12 CXCL10 CXCL16 F11R IL19 IL1RN IL5 LEFTY2 NR3C1 PTPRA FN1

-1 1 3 5

Fold Change Log 2 (2 -∆∆ C t )

Figure 10

Real time analysis of the gene expression of chemokines after treatment with Thymosin alpha 1 in PBMCs from HIV+ patients (a) and healthy donors (b).

a) HIV+

b) HIV-

MC P -1 MI P 1 -a lp h a Ra n te s PA R C MD C L D 78 be ta CCL 4 L 1 CX CL 1 6

-3 -1 1 3

genes

MC P -1 M IP1 -a lpha Rant es PA RC MD C LD7 8 be ta CCL4L1 CXC L 1 6

-3 -1 1 3

genes

*

*

* *

Fold Change Log 2 (2 -∆∆ C t )

*

*

*

*

*

*

CSF1R CSF2 CSF3R LTB MYD88 TLR2 MIF

-3 -1 1 3

genes

CSF1R CSF2 CSF3R LTB MYD88 TLR2 MIF

-3 -1 1 3

genes

a) HIV+

b) HIV-

Figure 11

Real time analysis of gene involved in Inflammatory response and in

cytokine-cytokine receptors interaction after treatment with Thymosin

alpha 1 in PBMCs from HIV+ patients (a) and healthy donors (b).

-5 -4 -3 -2 -1 0 1 2

CTR Tα1 -5

-4 -3 -2 -1 0 1 2

CTR Tα1

-5 -4 -3 -2 -1 0 1

2 ^{* p=0,021}

CTR Tα1

-5 -4 -3 -2 -1 0 1

2 ^{* p=0,019}

CTR Tα1

CCL4L1

CSF3R CXCL16

LTB

Log 2 (2 -∆ C t )

Figure 12a

Real time analysis of gene expression after treatment with Thymosin alpha 1 in PBMCs from HIV+ patients and healthy donors; values are normalized for the housekeeping gene and expressed as Log

(2

)

HIV - HIV +

-5 -4 -3 -2 -1 0 1 2

3 ^{* p=0,017}

CTR Tα1

PARC

-5 -4 -3 -2 -1 0 1

2

CTR Tα1

-8 -7 -6 -5 -4 -3 -2 -1 0 1

2 ^{* p=0,016}

CTR Tα1

-5 -4 -3 -2 -1 0 1 2

3 ^{* p=0,023}

CTR Ta1

Log 2 (2 -∆ C t )

Figure 12b

Real time analysis of gene expression after treatment with Thymosin alpha 1 in PBMCs from HIV+ patients and healthy donors; values are normalized for the housekeeping gene and expressed as Log

(2

)

MDC MIF

TLR2

HIV -

HIV +