Decreased memory T-cell response and function in human immunodeficiency virus-infected patients with tegumentary leishmaniasis

Gois, Luana Leandro; Mehta, Sanjay; Rodrigues, Maria Zilma Andrade; Schooley, Robert T; Badaró, Roberto; Grassi, Maria Fernanda Rios

doi:10.1590/0074-0276130174

Abstract

The effects of human immunodeficiency virus (HIV) on the immune response in patients with cutaneous leishmaniasis have not yet been fully delineated. This study quantified and evaluated the function of memory T-cell subsets in response to soluble Leishmania antigens (SLA) from patients coinfected with HIV and Leishmania with tegumentary leishmaniasis (TL). Eight TL/HIV coinfected subjects and 10 HIV seronegative subjects with TL were evaluated. The proliferative response of CD4+and CD8+T-cells and naïve, central memory (CM) and effector memory (EM) CD4+T-cells in response to SLA were quantified using flow cytometry. The median cell division indices for CD4+and CD8+T-cells of coinfected patients in response to SLA were significantly lower than those in patients with Leishmania monoinfection (p < 0.05). The proportions of CM and EM CD4+T-cells in response to SLA were similar between the coinfected patients and patients with Leishmania monoinfection. However, the median CM and EM CD4+T-cell counts from coinfected patients were significantly lower (p < 0.05). The reduction in the lymphoproliferative response to Leishmaniaantigens coincides with the decrease in the absolute numbers of both EM and CM CD4+T-cells in response to Leishmania antigens in patients coinfected with HIV/Leishmania.

Leishmania ; HIV; coinfection; memory CD4⁺ T-cells

Human immunodeficiency virus (HIV) type 1 is a human retrovirus that infects roughly 34 million people worldwide and is responsible for the majority of the acquired immune deficiency syndrome (AIDS) epidemic. The most severely affected areas are in Sub-Saharan Africa, the Caribbean, Eastern Europe and Central Asia (UNAIDS 2012). Leishmania have been recognised as opportunistic pathogens in immunocompromised individuals, including those infected with HIV (Badaro 1997, Cruz et al. 2006). Over the past decades, the HIV epidemic has spread from urban areas to suburban and rural areas, where leishmaniasis is found. In particular, Leishmania and HIV infection overlap in several sub-tropical and tropical regions around the world (Karp & Auwaerter 2007). In northern India, Sudan and Ethiopia, visceral leishmaniasis has been a major scourge in patients infected with HIV (Wolday et al. 2001, Mathur et al. 2006, ter Horst et al. 2008). In Brazil, cutaneous disease is also a common presentation of leishmaniasis in HIV-coinfected patients (Rabello et al. 2003, Carranza-Tamayo et al. 2009, Lindoso et al. 2009). These patients often present with multiple skin ulcers, disseminated lesions and nasopharyngeal and/or genital mucosal damage (Coura et al. 1987, Machado et al. 1992, Da-Cruz et al. 1999, Lindoso et al. 2009). The clinical outcome of Leishmania infection is dependent upon the balance of T-helper (Th)1 and Th2 cytokine production during the immune response (Bacellar et al. 2000, Bourreau et al. 2003). In HIV/Leishmania coinfected patients with visceral disease, HIV infection alters the Th1/Th2 balance, resulting in an increased Th2 cytokine response while levels of the Th1 cytokines interleukin (IL)-12 and IL-18 are decreased (Nigro et al. 1999, Wolday et al. 2000). This results in more aggressive and relapsing visceral leishmaniasis. The effects of HIV on the immune response in cutaneous leishmaniasis have not yet been fully delineated. Reduced interferon (IFN)-γ production, decreased IFN-γ/IL-10 ratios (Rodrigues et al. 2011) and diminished lymphoproliferative responses to Leishmania antigens have been described in HIV-infected patients with the cutaneous form of leishmaniasis (Da-Cruz et al. 1992, 1999). These findings suggest that coinfected HIV/Leishmania patients may have a memory T-cell defect. Here, we have quantified and assessed the function of memory T-cell subsets in response to soluble Leishmania antigens (SLA) from patients coinfected with HIV and Leishmania with tegumentary leishmaniasis (TL).

SUBJECTS, MATERIALS AND METHODS

Eight patients with TL and HIV infection and 10 HIV seronegative subjects with TL were included in the study. The subjects were enrolled from the AIDS clinical centre of the University Hospital Professor Edgard Santos, located in Salvador, Brazil, after informed consent. Clinical and demographic characteristics of the HIV/Leishmaniacoinfected subjects are presented in Table I. All Leishmania monoinfected patients had active cutaneous leishmaniasis at the time of inclusion in the study. Six of these patients had cutaneous leishmaniasis, one had disseminated cutaneous leishmaniasis and three had mucosal leishmaniasis.

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TABLE 1
Clinical and epidemiological characteristics of human immunodeficiency virus (HIV)/Leishmania coinfected patients

Peripheral blood mononuclear cells (PBMCs) were isolated by passage over a Ficoll-Hypaque gradient (Amersham Biosciences, Piscataway, NJ, USA). PBMCs (3 x 10⁶ cells) were resuspended in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2 mM L-glutamine, penicillin (100 U/mL), streptomycin (100 mg/mL) (GIBCO) and 5% heat-inactivated human AB serum. The cells were stimulated with SLA obtained from the Leishmania chagasi (MHOM/BR2000/Merivaldo2) strain [isolated from a patient with visceral leishmaniasis (Baleeiro et al. 2006)] or phytohemagglutinin (PHA) (Sigma-Aldrich). Control cultures were also established with medium alone.

To evaluate the proliferative response of T-cell subsets to SLA, 1.5 x 10⁶PBMC/mL were labelled with carboxyfluorescein succinimidyl ester (Invitrogen, Eugene, OR, USA) as described by the manufacturer. PBMCs were cultured in 12 × 75-mm tissue culture tubes for five days at 37ºC in a humidified 5% CO₂ atmosphere. Then, the cells were washed with phosphate-buffered saline (PBS)/bovine serum albumin and surface stained with anti-CD4-phycoerythrin (PE) (Becton Dickinson, CA, USA) and anti-CD8-PE-Cy5 (eBioscience, CA, USA) for 30 min at 4ºC. After fixation in 1% formaldehyde, the cells were acquired by FACSAria (Becton Dickinson) and analysis was performed using FlowJo software version 7.6 (Tree Star Inc, Ashland, OR, USA). At least 30,000 events were analysed per sample. The proliferation intensity was determined through the percentage of the cell division index. The threshold to define positive proliferative response was a cell division index above 0.06 for CD4⁺ T-cells and above 0.09 for the CD8⁺ T-cell subset as described previously (Pinto et al. 2011).

Alternatively, to determine the frequency of memory CD4⁺ T-cells, 1.5 x 10⁶ PBMCs were cultured with SLA, PHA or PBS for 18 h in supplemented RPMI-1640 medium in the presence of 0.5 µg of purified anti-CD28 antibody at 37ºC in a humidified 5% CO2 atmosphere. Then, the cells were stained with anti-CD4-(fluorescein isothiocyanate), anti-CD45RA-PE, anti-CD62L-PE-Cy5 and anti-CCR7-PE-Cy7 antibodies (Becton Dickinson). Cells were acquired by FACSAria and analysed using the Flowjo software. Naïve cells were defined as CD4⁺CD45RA⁺CD62L⁺CCR7⁺, central memory (CM) cells were defined as CD4⁺CD45RA-CD62L⁺CCR7⁺ and effector memory (EM) cells were defined as CD4⁺CD45RA-CD62L-CCR7-. Absolute naïve, CM and EM lymphocyte subpopulation counts were obtained by multiplying the subpopulation percentage by the absolute lymphocyte count.

The data are expressed as median and interquartile range (IQR). Statistical analysis was performed using GraphPad Prism 5.02 (La Jolla, CA, USA). The Mann-Whitney U test was used for group comparisons and the Wilcoxon test was used for the analyses of cell count and cell proportion in the presence and absence of antigen.

Institutional review board approvals were obtained from Brazilian National Ethical Commission, Oswaldo Cruz Foundation and the University of California San Diego Human Research Protection Program.

RESULTS

The median CD4⁺ T-cell count from HIV/Leishmania coinfected patients (241 cells/mm³, IQR 85-453) was significantly lower compared with that of Leishmania monoinfected patients (784 cells/mm³, IQR 599-1477) (p = 0.004). The median HIV viral load of HIV/Leishmaniacoinfected patients was 4.6 log copies/mL (IQR 1.9-5.2). The sex ratio and median age were similar in both groups.

The median cell division indices in response to SLA for CD4⁺ (0.06, IQR 0.05-0.1) and CD8⁺ T-cells (0.05, IQR 0.01-0.08) from HIV/Leishmania coinfected patients were significantly lower than those from Leishmania monoinfected patients (CD4⁺ T-cell: 0.18, IQR 0.15-0.26; CD8⁺ T-cell: 0.2, IQR 0.15-0.26) (p < 0.05) (Figure). A lymphoproliferative response to SLA was detected in only one coinfected patient (TL/HIV 06) (12%) who had a previously treated cutaneous leishmaniasis infection. In contrast, CD4⁺ and CD8⁺T-cell subsets from all Leishmania monoinfected patients proliferated in response to SLA. The CD4⁺ and CD8⁺ T-cell subsets from both HIV/Leishmania coinfected and Leishmaniamonoinfected patients all proliferated in response to PHA.

The proportions of naïve, CM and EM CD4⁺ T-cells obtained after stimulation with SLA were similar between the HIV/Leishmania coinfected and Leishmania monoinfected groups (Table II). However, the median absolute counts of naïve, CM and EM CD4⁺ T-cells from HIV/Leishmania coinfected patients were significantly lower compared with those of Leishmania monoinfected patients (p < 0.05). Following SLA stimulation, a statistically significant increase in the median absolute counts of EM CD4⁺ T-cells was observed in the cultures from Leishmania monoinfected patients (p = 0.005). In contrast, there was no change in the median absolute counts of EM CD4⁺ T-cells of HIV/Leishmania coinfected patients in the presence of SLA and a statistically significant decrease in the CM CD4⁺ T-cell count was observed after culture with SLA (p = 0.02). However, there was a statistically significant increase in the absolute number of EM CD4⁺ T-cells from both HIV/Leishmania coinfected and Leishmaniamonoinfected groups following PHA stimulation.

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TABLE II
Quantification of naïve, central (CM) and effector (EM) memory CD4+ T-cell subsets in response to soluble Leishmania antigens (SLA) and to phytohaemagglutinin (PHA) from of human immunodeficiency virus (HIV)/Leishmania coinfected and Leishmania monoinfected groups

Wilcoxon test was used to compare cells cultured with medium and SLA (a) and cells cultured with medium and PHA (b) for each group. Data are presented as median (interquartile range). p: Mann-Whitney U test was used to compare HIV/Leishmaniacoinfected and Leishmaniasis monoinfected groups.

DISCUSSION

This study demonstrates that a reduction in the lymphoproliferative response to Leishmania antigens coincides with a decrease in the absolute numbers of both EM and CM CD4⁺ T-cells in response to Leishmania antigens in patients coinfected with HIV/Leishmania. Although the proportion of memory CD4⁺T-cells subsets was similar between monoinfected Leishmania and coinfected HIV/Leishmania patients, the median absolute count of EM lymphocytes in coinfected patients was four-fold lower compared with that of Leishmania monoinfected patients. Moreover, following Leishmania antigen stimulus, the EM CD4⁺ T-cell count of HIV/Leishmania coinfected patients remained below 10 cells/mm³, whereas we observed a three-fold increase in the EM CD4⁺ T-cell count in the Leishmania monoinfected patients. The low number of EM CD4⁺ T-cells in HIV/Leishmania coinfected patients might explain the reduced IFN-γ production in response to Leishmania antigens observed in coinfected subjects as well as the atypical parasitic spread, frequent relapse and treatment failure observed in these patients (Da-Cruz et al. 1992, Cruz et al. 2006, Lindoso et al. 2009). Although memory and effector CD8⁺ T-cell profiles were not evaluated in our study, CD8⁺T-cells from all HIV/Leishmania coinfected patients were unable to proliferate in response to SLA. Previous work has demonstrated the protective role of CD8⁺ T-cells in Leishmania infection, with healing of lesions associated with high IFN-γ production and an increase in the number of CD8⁺ T-cells (Da-Cruz et al. 1994, Muller et al. 1994).

The absence of a lymphoproliferative response to Leishmania antigens in both the CD4⁺ and CD8⁺ T-cell subsets examined in our study suggests impairment of the cellular immune response directed at the parasitic infection. Although the HIV/Leishmania coinfected group was heterogeneous with respect to clinical presentation of leishmaniasis (cutaneous vs. mucosal) and stage of treatment (active vs. treated), a significant lack of immune response to Leishmania antigens was observed across this group of subjects. Only one HIV/Leishmania coinfected patient exhibited a proliferative response to Leishmania antigens. This patient was successfully treated for TL before his inclusion in the study and his lesions were in remission. However, the two other coinfected individuals with treated TL did not develop a proliferative response. Additionally, we found that a decrease in the number of CM CD4⁺T-cells occurred following SLA stimulation in all cultures of coinfected patients and EM CD4⁺ T-cell counts of these patients did not change regardless of presentation of leishmaniasis or stage of treatment. Previous work has shown that the restoration of CD4⁺ T-cell-specific responses to antigens in HIV-infected patients undergoing antiretroviral treatment (ART) requires a sustained increase of at least 60 CD4⁺ T-cells/µL compared with baseline levels and a reduced viral load (Li et al. 1998). In addition to the quantitative immune defect observed in HIV infection, the quality of the immune response (i.e., degree of immune memory impairment) may also be important in the host response to other pathogens such as Leishmania.

Studies in both mice and humans have demonstrated multiple CD4⁺ T-cell phenotypes and a broad spectrum of functions have been attributed to these cells in different viral infections (Chen et al. 2001, de Rosa et al. 2001, Roman et al. 2002). Memory T-cells are divided into subpopulations based on the expression of CCR7, a chemokine receptor involved in homing to secondary lymphoid organs and CD45RA, a cell surface molecule involved in T-cell activation. T-cells expressing CD45RA and CCR7 are termed naïve, whereas those with downregulated CD45RA are termed CM cells and produce IL-2. Memory T-cells lacking expression of CCR7 and CD45RA are termed EM T-cells and produce IFN-γ (Sallusto et al. 1999). The functional and phenotypic heterogeneity of memory T-cells are influenced by variations in antigen exposure and persistence. A dominant single IL-2 and/or IL-2/IFN-γ CD4 T-cell response is normally associated with clearance of antigen, whereas a dominant single IFN-γ CD4 T-cell response is associated with antigen persistence and high antigen levels (Harari et al. 2005). Effector CD4⁺ T-cells are lost in the absence of parasites, whereas CM CD4⁺ T-cells are maintained. Upon secondary infection, these CM T-cells become effector T-cells and mediate protection (Zaph et al. 2004). In our study, a decrease in the number of CM CD4⁺ T-cells was observed in the coinfected group following stimulation with SLA. It is possible that during coinfection with HIV and Leishmania, the immune activation caused by both HIV and Leishmania infection induces increased apoptosis of parasite-specific memory CD4⁺ T-lymphocytes (Wolday et al. 1999a, b). The decreased numbers of CM cells and EM cells would reduce the ability of the immune system to develop an adequate systemic response to control the Leishmaniainfection. However, the overall hyporesponsiveness of the cells to Leishmania antigen stimulation could also suggest that a qualitative defect may be contributing to this phenomenon. Thus, although an impaired memory response is known to be related to the depletion of CD4⁺ T-cells via mechanisms related directly or indirectly to HIV (systemic activation and replicative senescence), a defective functional memory response may also be playing a role (Moro-Garcia et al. 2012). Interestingly however, we found that our coinfected cells responded well to PHA, suggesting that the immunodeficiency was parasite-specific.

Viral suppression by ART may be a contributing factor and in this study, several of our subjects were not receiving ART. In the future, we will examine how the immune response to Leishmania is reconstituted under ART. Although the impact of HIV-1 on host cellular immune function clearly plays an important role in facilitating Leishmania infection in dually infected individuals, there is increasing evidence that other mechanisms for the interaction of these two pathogens might also be involved. Particularly, the immunological mechanisms involved in the pathogenesis of mucocutaneous lesions in HIV-infected individuals remain unclear. We hypothesise that innate immunity to Leishmania is also compromised in coinfected patients, resulting in the more severe pathogenesis of mucocutaneous lesions observed in coinfected patients. Future work will be required to evaluate the effect of coinfection on innate immunity.

To Geraldo Gileno, LPBI, Fiocruz-BA, for providing SLA.

REFERENCES

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Badaro R 1997. When Leishmania and HIV interact, a new broad spectrum of leishmaniasis occurs. Braz J Infect Dis 1: 145-148.
Baleeiro CO, Paranhos-Silva M, dos Santos JC, Oliveira GG, Nascimento EG, de Carvalho LP, dos Santos WL 2006. Montenegro's skin reactions and antibodies against different Leishmania species in dogs from a visceral leishmaniosis endemic area. Vet Parasitol 139: 21-28.
Bourreau E, Gardon J, Pradinaud R, Pascalis H, Prevot-Linguet G, Kariminia A, Pascal L 2003. Th2 responses predominate during the early phases of infection in patients with localized cutaneous leishmaniasis and precede the development of Th1 responses. Infect Immun 71: 2244-2246.
Carranza-Tamayo CO, de Assis TS, Neri AT, Cupolillo E, Rabello A, Romero GA 2009. Prevalence of Leishmania infection in adult HIV/AIDS patients treated in a tertiary-level care center in Brasilia, Federal District, Brazil. Trans R Soc Trop Med Hyg 103: 743-748.
Chen G, Shankar P, Lange C, Valdez H, Skolnik PR, Wu L, Manjunath N, Lieberman J 2001. CD8 T cells specific for human immunodeficiency virus, Epstein-Barr virus and cytomegalovirus lack molecules for homing to lymphoid sites of infection. Blood 98: 156-164.
Coura JR, Galvão-Castro B, Grimaldi Jr G 1987. Disseminated American cutaneous leishmaniasis in a patient with AIDS. Mem Inst Oswaldo Cruz 82: 581-582.
Cruz I, Nieto J, Moreno J, Canavate C, Desjeux P, Alvar J 2006. Leishmania/HIV co-infections in the second decade. Indian J Med Res123: 357-388.
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Da-Cruz AM, Machado ES, Menezes JA, Rutowitsch MS, Coutinho SG 1992. Cellular and humoral immune responses of a patient with American cutaneous leishmaniasis and AIDS. Trans R Soc Trop Med Hyg 86: 511-512.
de Rosa SC, Herzenberg LA, Roederer M 2001. 11-color, 13-parameter flow cytometry: identification of human naïve T cells by phenotype, function and T-cell receptor diversity. Nat Med 7: 245-248.
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Karp CL, Auwaerter PG 2007. Coinfection with HIV and tropical infectious diseases. I. Protozoal pathogens. Clin Infect Dis 45: 1208-1213.
Li TS, Tubiana R, Katlama C, Calvez V, Mohand HA, Autran B 1998. Long-lasting recovery in CD4 T-cell function and viral-load reduction after highly active antiretroviral therapy in advanced HIV-1 disease. Lancet 351: 1682-1686.
Lindoso JA, Barbosa RN, Posada-Vergara MP, Duarte MI, Oyafuso LK, Amato VS, Goto H 2009. Unusual manifestations of tegumentary leishmaniasis in AIDS patients from the New World. Br J Dermatol 160: 311-318.
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Mathur P, Samantaray JC, Vajpayee M, Samanta P 2006. Visceral leishmaniasis/human immunodeficiency virus co-infection in India: the focus of two epidemics. J Med Microbiol 55: 919-922.
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Rodrigues MZ, Grassi MF, Mehta S, Zhang XQ, Gois LL, Schooley RT, Badaro R 2011. Th1/Th2 cytokine profile in patients coinfected with HIV and Leishmania in Brazil. Clin Vaccine Immunol 18: 1765-1769.
Roman E, Miller E, Harmsen A, Wiley J, von Andrian UH, Huston G, Swain SL 2002. CD4 effector T cell subsets in the response to influenza: heterogeneity, migration and function. J Exp Med 196: 957-968.
Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401: 708-712.
ter Horst R, Collin SM, Ritmeijer K, Bogale A, Davidson RN 2008. Concordant HIV infection and visceral leishmaniasis in Ethiopia: the influence of antiretroviral treatment and other factors on outcome. Clin Infect Dis 46: 1702-1709.
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Publication Dates

Publication in this collection
02 Oct 2013
Date of issue
Feb 2014

History

Received
27 Mar 2013
Accepted
18 July 2013

[1] Bacellar O, D'Oliveira Jr A, Jeronimo S, Carvalho EM 2000. IL-10 and IL-12 are the main regulatory cytokines in visceral leishmaniasis. Cytokine 12: 1228-1231.

[2] Badaro R 1997. When Leishmania and HIV interact, a new broad spectrum of leishmaniasis occurs. Braz J Infect Dis 1: 145-148.

[3] Baleeiro CO, Paranhos-Silva M, dos Santos JC, Oliveira GG, Nascimento EG, de Carvalho LP, dos Santos WL 2006. Montenegro's skin reactions and antibodies against different Leishmania species in dogs from a visceral leishmaniosis endemic area. Vet Parasitol 139: 21-28.

[4] Bourreau E, Gardon J, Pradinaud R, Pascalis H, Prevot-Linguet G, Kariminia A, Pascal L 2003. Th2 responses predominate during the early phases of infection in patients with localized cutaneous leishmaniasis and precede the development of Th1 responses. Infect Immun 71: 2244-2246.

[5] Carranza-Tamayo CO, de Assis TS, Neri AT, Cupolillo E, Rabello A, Romero GA 2009. Prevalence of Leishmania infection in adult HIV/AIDS patients treated in a tertiary-level care center in Brasilia, Federal District, Brazil. Trans R Soc Trop Med Hyg 103: 743-748.

[6] Chen G, Shankar P, Lange C, Valdez H, Skolnik PR, Wu L, Manjunath N, Lieberman J 2001. CD8 T cells specific for human immunodeficiency virus, Epstein-Barr virus and cytomegalovirus lack molecules for homing to lymphoid sites of infection. Blood 98: 156-164.

[7] Coura JR, Galvão-Castro B, Grimaldi Jr G 1987. Disseminated American cutaneous leishmaniasis in a patient with AIDS. Mem Inst Oswaldo Cruz 82: 581-582.

[8] Cruz I, Nieto J, Moreno J, Canavate C, Desjeux P, Alvar J 2006. Leishmania/HIV co-infections in the second decade. Indian J Med Res123: 357-388.

[9] Da-Cruz AM, Conceicao-Silva F, Bertho AL, Coutinho SG 1994. Leishmania-reactive CD4+ and CD8+ T cells associated with cure of human cutaneous leishmaniasis. Infect Immun 62: 2614-2618.

[10] Da-Cruz AM, Filgueiras DV, Coutinho Z, Mayrink W, Grimaldi Jr G, De Luca PM, Mendonça SCF, Coutinho SG 1999. Atypical mucocutaneous leishmaniasis caused by Leishmania braziliensis in an acquired immunodeficiency syndrome patient: T-cell responses and remission of lesions associated with antigen immunotherapy. Mem Inst Oswaldo Cruz 94: 537-542.

[11] Da-Cruz AM, Machado ES, Menezes JA, Rutowitsch MS, Coutinho SG 1992. Cellular and humoral immune responses of a patient with American cutaneous leishmaniasis and AIDS. Trans R Soc Trop Med Hyg 86: 511-512.

[12] de Rosa SC, Herzenberg LA, Roederer M 2001. 11-color, 13-parameter flow cytometry: identification of human naïve T cells by phenotype, function and T-cell receptor diversity. Nat Med 7: 245-248.

[13] UNAIDS - Joint United Nations Program on HIV/AIDS 2012. Global Report 2012. UNAIDS report on the global AIDS epidemic, WHO, Geneva, 107 pp.

[14] Harari A, Vallelian F, Meylan PR, Pantaleo G 2005. Functional heterogeneity of memory CD4 T cell responses in different conditions of antigen exposure and persistence. J Immunol 174: 1037-1045.

[15] Karp CL, Auwaerter PG 2007. Coinfection with HIV and tropical infectious diseases. I. Protozoal pathogens. Clin Infect Dis 45: 1208-1213.

[16] Li TS, Tubiana R, Katlama C, Calvez V, Mohand HA, Autran B 1998. Long-lasting recovery in CD4 T-cell function and viral-load reduction after highly active antiretroviral therapy in advanced HIV-1 disease. Lancet 351: 1682-1686.

[17] Lindoso JA, Barbosa RN, Posada-Vergara MP, Duarte MI, Oyafuso LK, Amato VS, Goto H 2009. Unusual manifestations of tegumentary leishmaniasis in AIDS patients from the New World. Br J Dermatol 160: 311-318.

[18] Machado ES, Braga MP, Da-Cruz AM, Coutinho SG, Vieira ARM, Rutowitsch MS, Cuzzi-Maya T, Grimaldi Jr G, Menezes JA 1992. Disseminated American muco-cutaneous leishmaniasis caused by Leishmania braziliensis braziliensis in a patient with AIDS: a case report. Mem Inst Oswaldo Cruz 87: 487-492.

[19] Mathur P, Samantaray JC, Vajpayee M, Samanta P 2006. Visceral leishmaniasis/human immunodeficiency virus co-infection in India: the focus of two epidemics. J Med Microbiol 55: 919-922.

[20] Moro-Garcia MA, Alonso-Arias R, Lopez-Larrea C 2012. Molecular mechanisms involved in the aging of the T-cell immune response. Curr Genomics 13: 589-602.

[21] Muller I, Kropf P, Louis JA, Milon G 1994. Expansion of gamma interferon-producing CD8+ T cells following secondary infection of mice immune to Leishmania major. Infect Immun 62: 2575-2581.

[22] Nigro L, Cacopardo B, Preiser W, Braner J, Cinatl J, Palermo F, Russo R, Doerr HW, Nunnari A 1999. In vitro production of type 1 and type 2 cytokines by peripheral blood mononuclear cells from subjects coinfected with human immunodeficiency virus and Leishmania infantum. Am J Trop Med Hyg60: 142-145.

[23] Pinto LA, Castro BG, Soares MBP, Grassi MFR 2011. An evaluation of the spontaneous proliferation of peripheral blood mononuclear cells in HTLV-1-infected individuals using flow cytometry. ISRN Oncol 2011: 326719.

[24] Rabello A, Orsini M, Disch J 2003. Leishmania/HIV co-infection in Brazil: an appraisal. Ann Trop Med Parasitol 97 (Suppl. 1): 17-28.

[25] Rodrigues MZ, Grassi MF, Mehta S, Zhang XQ, Gois LL, Schooley RT, Badaro R 2011. Th1/Th2 cytokine profile in patients coinfected with HIV and Leishmania in Brazil. Clin Vaccine Immunol 18: 1765-1769.

[26] Roman E, Miller E, Harmsen A, Wiley J, von Andrian UH, Huston G, Swain SL 2002. CD4 effector T cell subsets in the response to influenza: heterogeneity, migration and function. J Exp Med 196: 957-968.

[27] Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401: 708-712.

[28] ter Horst R, Collin SM, Ritmeijer K, Bogale A, Davidson RN 2008. Concordant HIV infection and visceral leishmaniasis in Ethiopia: the influence of antiretroviral treatment and other factors on outcome. Clin Infect Dis 46: 1702-1709.

[29] Wolday D, Akuffo H, Demissie A, Britton S 1999a. Role of Leishmania donovani and its lipophosphoglycan in CD4+ T-cell activation-induced human immunodeficiency virus replication. Infect Immun 67: 5258-5264.

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Coinfected group	Gender	Age (year)	CD4⁺ T-cell count (cell/mm³)	Viral load (log copies/mL)	ART use	Leishmaniasis form

TL/HIV 03	F	42	93	5.4	Unknown	Mucocutaneous active
TL/HIV 05	F	22	291	1.9	Unknown	Cutaneous active
TL/HIV 06	M	36	165	5.3	Unknown	Cutaneous treated
TL/HIV 08	F	62	415	2.7	Yes	Disseminated cutaneous treated
TL/HIV 09	M	59	78	5.0	Yes	Cutaneous active
TL/HIV 12	M	36	615	1.9	Yes	Cutaneous active
TL/HIV 13	M	36	104	5.0	No	Cutaneous active treated
TL/HIV 14	M	25	241	4.6	Yes	Mucocutaneous active

CD4⁺ T-cell subset	Medium			SLA			PHA
CD4⁺ T-cell subset	Coinfected	Monoinfected	p	Coinfected	Monoinfected	p	Coinfected	Monoinfected	p

Naïve
n (%)	30 (17-37)	43 (18-80)	0.8	39 (17-64)	34 (15-57)	0.7	2 (1-4)^b	4 (2-9)^b	0.5
n (cell/mm³)	9 (3-14)	17 (5-65)	0.02	3 (2-11)	42 (25-106)	0.03	2 (0-6)^b	13 (4-28)	0.02
CM
n (%)	23 (8-31)	24 (6-32)	0.6	11 (4-25)	8 (2-23)^a	0.9	1 (0-4)^b	2 (1-3)	0.3
n (cell/mm³)	55 (31-105)	49 (19-103)	0.001	3 (1-5) ^a	46 (25-119)	0.8	1 (0-3)^b	8 (2-16)^b	0.02
EM
n (%)	12 (11-29)	9 (6-15)	0.2	21 (11-43)	17 (14-23)^a	0.07	35 (20-40)^b	30 (25-38)^b	0.8
n (cell/mm³)	8 (5-11)	36 (12-63)	0.0009	6 (2-9)	122 (69-143)^a	0.0001	27 (11-58)^b	175 (105-236)^b	0.0005