Enhanced Lesional Foxp3 Expression and Peripheral Anergic Lymphocytes Indicate a Role for Regulatory T Cells in Indian Post-Kala-Azar Dermal Leishmaniasis

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Enhanced Lesional Foxp3 Expression and Peripheral Anergic Lymphocytes Indicate a Role for Regulatory T Cells in Indian Post-Kala-Azar Dermal Leishmaniasis Sudipto Ganguly, Debanjan Mukhopadhyay, Nilay K. Das, Mehervani Chaduvula, Soumi Sadhu, Uttara Chatterjee, Mehebubar Rahman, Rama P. Goswami, Subhashish Kamal Guha, Dolanchampa Modak, Sudeshna Mallik, Debananda Gonju, Netai Pramanik, Joyashree N. Barbhuiya, Bibhuti Saha, Mitali Chatterjee Journal of Investigative Dermatology Volume 130, Issue 4, Pages 1013-1022 (April 2010) DOI: 10.1038/jid.2009.393 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Specific and generalized proliferative responses of lymphocytes. Lymphocytes from patients with post-kala-azar dermal leishmaniasis (PKDL) before (▪, n=21) and after (□, n=12) treatment, from patients with visceral leishmaniasis (VL) after cure (, n=5), and from healthy controls (, n=6) were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE), incubated with/without Leishmania donovani antigen (LDA; 5μgml–1, a) or phytohemagglutinin (PHA; 10μgml–1, b), and CFSE fluorescence of gated CD3+CD4+ and CD3+CD8+ lymphoblasts was determined using flow cytometry. Columns represent mean proliferation indices of at least five individuals per group, with error bars denoting SEM. *P<0.05, **P<0.01, significantly different from controls; @P<0.05, significantly different from VL after treatment. (c) Representative profile of proliferative responses of CD3+CD4+ and CD3+CD8+ lymphocytes to LDA in patients with PKDL before and after treatment, cured VL patients, and healthy individuals. (d) Representative profile of proliferative responses of CD3+CD4+ and CD3+CD8+ lymphocytes to PHA in patients with PKDL before and after treatment, cured VL patients, and healthy individuals. Journal of Investigative Dermatology 2010 130, 1013-1022DOI: (10.1038/jid.2009.393) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Longitudinal monitoring of lesional expression of IFN-γ and IL-10 in patients with post-kala-azar dermal leishmaniasis (PKDL) before and after treatment. (a) Representative profile of mRNA expression of IFN-γ and IL-10 in lesional tissue from patients with PKDL (n=5) before and after treatment and normal skin tissue from healthy individuals (n=3). Isolated RNA was subjected to reverse transcriptase-PCR (RT-PCR); products were resolved using agarose gel electrophoresis with bands specific for β-actin, IFN-γ, and IL-10, visualized using UV transillumination, and quantified using densitometric analysis, as described in Materials and Methods. (b) Before–after plots of IFN- γ and IL-10 expression in patients with PKDL (n=12) before (▴) and after completion of treatment (▵). (c) Scatter plots of expression values for IFN-γ and IL-10 in patients with PKDL (n=12) before (▴) and after treatment (▵) as well as in healthy controls (⧫, n=3); horizontal lines indicate mean values. *P<0.01, **P<0.001, significantly different from controls; @P<0.001, significantly different from pre-treatment. Journal of Investigative Dermatology 2010 130, 1013-1022DOI: (10.1038/jid.2009.393) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Lesional forkhead box protein 3 (Foxp3) expression in patients with post-kala-azar dermal leishmaniasis (PKDL) at presentation and after treatment. (a) A representative profile of Foxp3 mRNA expression in lesional tissue from patients with PKDL (n=5) before and after treatment and normal skin tissue from three healthy individuals. Expression was quantified using reverse transcriptase-PCR (RT-PCR) and subsequent densitometric analysis, as described in Materials and Methods. (b) Before–after plot of Foxp3 expression in patients with PKDL (n=12) before (▴) and after completion of treatment (▵). (c) Scatter plot showing Foxp3 expression values in patients with PKDL (n=12) before (▴) and after treatment (▵) and in healthy controls (⧫, n=3). Horizontal lines indicate mean values. *P<0.01, significantly different from controls. Journal of Investigative Dermatology 2010 130, 1013-1022DOI: (10.1038/jid.2009.393) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunohistochemistry of lesional sections from patients with post-kala-azar dermal leishmaniasis (PKDL). (a) A representative profile of CD3 and forkhead box protein 3 (Foxp3; 10–20% positivity) staining in lesional tissue sections from a patient with PKDL before and after treatment. (b) A representative profile of CD3 and Foxp3 (1–5% positivity) staining in lesional tissue sections from a patient with PKDL before and after treatment. Arrows indicate Foxp3-positive cells within the lymphocytic infiltrate. Bars=10μM. Journal of Investigative Dermatology 2010 130, 1013-1022DOI: (10.1038/jid.2009.393) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions