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Experimental Parasitology 118 (2008) 238–246

Leishmania donovani: Role of CD2 on CD4+ T-cell functionin Visceral Leishmaniasis

Sanjiva Bimal a,*, Shubhankar K. Singh a, Sukrat Sinha a, Krishna Pandey a,Prabhat K. Sinha a, Alok Ranjan a, Sujit K. Bhattacharya b, Pradeep Das a

a Division of Immunology, Rajendra Memorial Research Institute of Medical Sciences (ICMR), Agamkuan, Patna 800007, Indiab Indian Council of Medical Research, New Delhi, India

Received 13 November 2006; received in revised form 14 August 2007; accepted 15 August 2007Available online 22 August 2007

Abstract

In this study, we investigated whether alteration in the CD2 mediated coordination of an immune response was associated with downregulation of CD4 associated Th1 cell response during Visceral Leishmaniasis (VL). Leishmania donovani (Ld) infection in VL patientsmarkedly reduced expression of CD2 cell surface antigen on CD4+ cells. T-cells of VL patients were mostly in G0/G1 stage of the cellcycle (98.20%) with little or no activity of protein kinase C-a (PKC-a) isoform. However, pre-incubation with activating anti-CD2 mono-clonal antibody (MAb) resulted in a corresponding increase up to 2.52-fold in T-cells of G2/M population supported by both activity andexpression of PKC-a isoform. Furthermore, we observed that co-incubation of T-cell with anti-CD2 increased the lymphocyte-blast pop-ulation in patients in whom the CD4 cells became more antigen responsive (CD4+ CD69+ cells). Consistent with these observations, itwas shown that 59.3% of CD4 cells from patients responded to Ld by producing IFN-c. Even in the culture condition, when the T-cellsfrom patients were depleted of APC, IFN-c production was noticed after CD2 activation. On the other hand, IL-4 production becamelow in the anti-CD2 antibody supplemented peripheral blood mononuclear cells (PBMNCs) culture. These findings imply that infectionwith L. donovani induces less CD2 on the surface of CD4+ T-cells, which once activated orchestrate the protective IFN-c dominant hostdefense mechanism via PKC-mediated signal transduction and cell cycle.� 2007 Elsevier Inc. All rights reserved.

Index Descriptors and Abbreviations: VL, Visceral Leishmaniasis; Ld, Leishmania donovani; PBMNCs, peripheral blood mononuclear cells; APC, antigenpresenting cells; anti-CD2, anti-CD2 antibody; anti-PKC-a, anti-protein kinase C-a; IFN-c, interferon-gamma; IL-10, interleukin-10; IL-4, interleukin-4;r-IL-4, recombinant interleukin-4; IL-12, interleukin-12; PBS, phosphate buffered saline; FCS, fetal calf serum

1. Introduction

Visceral Leishmaniasis (VL) caused by Leishmania dono-

vani (Ld) in India is one of the several clinically importantinfections where Th1 sub-populations of CD4 cells fail toproduce interferon-gamma (IFN-c) which activate macro-phages and coordinate the immune response to intracellu-lar Leishmania species. On the other hand, expansion ofan interleukin-4 (IL-4) and interleukin-10 (IL-10)-biasedTh2 response inhibits the immune control in vivo and leads

0014-4894/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.exppara.2007.08.009

* Corresponding author. Fax: +91 0612 2634379.E-mail address: drsbimal@yahoo.com (S. Bimal).

to severe disseminated forms of the disease (Ghalib et al.,1993; Babaloo et al., 2001; Uzonna and Bretscher, 2001).Cell surface adhesion molecules of the immunoglobulinsuper-family-lymphocyte function associated antigen-1(LFA-1), CD2 and intracellular activation motifs(ICAM-3) can mediate T-cell adhesive interactions whichis later extended, through support of co-stimulatory recep-tor CD28 on T-cells, to enable an immune response tooccur (Janeway, 1992; Moingeon et al., 1989; Harriset al., 2000; Pulendran et al., 2001). As a result, antigenresponsive lymphocytes are shown to express CD69, anactivation marker during in vitro antigen stimulation (Sch-lossman et al., 1995). A study by Gallob et al. (1996) doc-umented a key role of CD2 antigen on T-cells in mediating

S. Bimal et al. / Experimental Parasitology 118 (2008) 238–246 239

the ability of monocytes to enhance T-cell activation by IL-12 during the ongoing process of antigen presentation byAPCs. Several studies have also documented the role ofCD2 on T-cells through thymic selection events and signaltransduction (Sasada and Reinherz, 2001; Yang and Rein-herz, 2001), which promotes proliferating T-cells to trans-duce efficient signaling to affect IFN-c production even inthe absence of APCs (Meuer et al., 1984; Green et al.,2000; Meinl et al., 2000; Sasada et al., 2002). The role ofIFN-c in forms of leishmaniasis is very important for resis-tance and cure and we previously reported a deficiency ofCD2 cell surface expression on T-cells in VL patients,which we presume might be a critical factor in activationof Th1 response of CD4+ T-cell during VL (Bimal et al.,2005). As the role of CD2 in infectious diseases has beenrelatively little studied, the present study was undertakento determine the immuno-modulatory effect of CD2 anti-gen co-receptor in the reversal of T-cell non-responsivenessto L. donovani antigen in acute Visceral Leishmaniasis.

2. Materials and methods

2.1. Subjects

Twenty subjects of both sexes aged between 18 and 27years (10 patients with acute VL in their pre-treatmentstage and 10 controls) were studied. Amastigotes of L.

donovani had been found in bone marrow and/or spleenaspirates from each of the VL cases investigated and, whenblood samples were collected, each of these cases hadsplenomegaly and had been suffering with continuous orintermittent fever for at least two weeks. The controlshad no splenomegaly or recent history of illness. Potentialsubjects who were unwilling to give their informed consentor were positive for tuberculosis (TB), kidney, heart andliver diseases, malaria and HIV were excluded.

2.2. Phenotype cell analysis

Peripheral blood mononuclear cells (PBMNCs) isolatedfrom blood samples by centrifugation (800g, 15 min) overHistopaque�-1077 (Sigma, USA) were washed in phos-phate buffered saline (PBS). Cells (1 · 106 in 50 ll) werestained with labeled antibodies specific for different humancell surface antigens (BD Pharmingen, San Diego, CA,USA) for 30 min, according to manufacturer’s instructions.The following monoclonal antibodies were used: FITClabeled anti-human CD2 (clone: RPA-2.10), FITC or PElabeled mouse anti-human CD3 (clone: UCHT1) or PElabeled mouse anti-human CD4 (clone: RPA-T4) or FITClabeled anti-human CD8 antibodies (clone: RPA-T8). Theun-reacted antibodies were removed by washing the cellstwice with PBS containing 2% FCS, pH 7.2 before eachsample was re-suspended in 450 ll stain buffer containing1% freshly prepared formaldehyde (MERCK BDH, India)for examination of fluorescence in a FACS-Calibur Flow-Cytometer (Beckton Dickinson, San Jose, CA, USA). Flow

data were evaluated on Cell Quest software. Negative con-trol samples were incubated with irrelevant isotypematched antibodies (FITC and PE labeled IgG; BDPharmingen, USA) in parallel with all experimentalsamples.

2.3. Cell cycle studies

Cycle TestTM plus DNA reagent kit (BD Pharmingen,San Diego, CA, USA) was used to compare the progres-sion of T-cells in the cell cycle with and without stimulationwith mouse anti-human-LFA-2 (anti-CD2), clone S5.2 (BDPharmingen, San Diego, CA, USA) after pre-incubation ofcells in L. donovani. In brief, the PBMNCs suspension(5 · 106 cells) was pulsed with L. donovani antigen (10lg/ml) and cultured in a water saturated air atmosphere with5% CO2 at 37 �C for 2 h. Later, T-cells were fractionatedfrom the PBMNCs suspension using nylon wool column.Following washing (400g, 5 min at room temperature) cellswere treated with 250 ll of a solution containing trypsin ina spermine tetrahydrochloride detergent buffer (kit content,BD Pharmingen, San Diego, CA, USA) for 10 min at RT todigest cell membrane and to stabilize the nuclear chromatin.The cells were later treated with 200 ll of a solution contain-ing trypsin inhibitor and RNase buffer (provided with thekit) for 10 min at RT. These cells were incubated with200 ll of propidium iodide (PI, provided with the kit) stainsolution for 10 min in the dark on ice to allow PI to boundto isolated nuclei. The stained cells were finally run on aFACS-Calibur (BD, San Diego, CA) with FL-2 detectorusing a 585/42-band pass filter. Finally, the FL-2-A DNAhistogram was analyzed using Modfit software (BectonDickinson) on the FACS-Calibur. An additional sampletube of PBMC mixed with experimental tubes was alsoprepared and used as a control.

2.4. Expression of PKC-a proteins in T-cells

Leishmania donovani primed PBMNCs (2 h) stimulatedin presence or absence of anti-CD2 (clone S5.2) for 16 h(as described above) were fixed with freshly prepared 2%formaldehyde (MERCK BDH, India) at 37 �C for10 min. Cells were washed and permeablized by cold 90%methanol on ice (30 min). After washing in stain buffer,we performed a co-staining of T-cells (1 · 106) using 20 llof anti-CD4 PE-conjugated antibody (BD Pharmingen,USA) and 1 lg of mouse IgG 2b anti-PKC-a antibody,Mol. Wt. 82 kDa, clone 3 (BD Pharmingen, USA) and20 ll mouse anti-human IgG antibodies conjugated toFITC (BD Pharmingen). Washed cells were re-suspendedin 500 ll of stain buffer and production of PKC-a byCD4+ T-cells of patients and control samples were deter-mined by flow cytometry. Further, cellular proteins of T-cells of patients and healthy control either stimulated ornot stimulated with anti-CD2 (unstimulated) were ana-lyzed by SDS–PAGE and Western blotting for the expres-sion of PKC-a following method as described by Laemmli

240 S. Bimal et al. / Experimental Parasitology 118 (2008) 238–246

(1970) and Towbin et al. (1979). Antibody used for PKC-awas against the phosphorylated form of this signalingmolecule.

2.5. Effect of CD2 on Leishmania specific activated T-cells

The PBMNC suspension was stimulated with fixed L.donovani antigen for 2 h and then cultured in fresh com-plete RPMI-1640 with 4 lg of mouse anti-human CD2(BD) for 16 h. These lymphocytes were analyzed for theLeishmania specific activation pattern after activation oftheir CD2 antigen by anti-CD2 antibody using the pro-gram ‘Cell Quest’ on FACS-Calibur. Firstly, the frequencyof blast cells was analyzed in forward and side scatter his-tograms through placement of R1 gate or small lympho-cytes (LC) and R2 gate or blast lymphocytes (LB).Further, to get a direct initial impact of CD2 on activationof CD4+ cells, cultured cells were washed and stained withmouse anti-human CD69-PE and mouse anti-human CD4-FITC (both from BD, USA) in a single staining step for15 min in dark that followed flow cytometry analysis.

2.6. Cytokine analysis

2.6.1. Fluorescence activated cell sorter (FACSTM) based

intracellular cytokine assay

Peripheral blood mononuclear cells (5 · 106/ml) werecultured in RPMI-1640 medium (Hi-media, India) supple-mented with 2 mM L-glutamine (Hi-media, India), 10%fetal calf serum (Life Technologies, Gaithersburg, MD),2 mM 2-mercaptoethanol (Sigma) and 50 lg/ml Gentamy-cin, 100 U/ml penicillin, 100 lg/ml Streptomycin and thenadjusted to pH 7.4 with 2 N NaOH. Cells were stimulatedin 24 well tissue culture plates with fixed (2% formalde-hyde) Ld promastigote antigen in responder (PBMNC) tostimulator (fixed Ld) ratio of 100:1 for 2 h at 37 �C andof them 5 · 106cells were cultured in 96 well round bot-tomed plates pre-coated with anti-CD2 (LFA-2) antibodies(4 lg/ml). Control cultures were set up in medium alone ormedium containing PHA 20 lg/ml. Intra-cytoplasmic cyto-kine level was detected on FACS-Calibur as previouslydescribed (Prussin and Metcalft, 1995; Litton et al.,1996). Cells (5 · 106) were cultured for 14 h followed by4 h incubation with a protein transport inhibitor, brefel-din-A (1 lg/ml). The harvested cells were consecutivelyco-incubated with PE-conjugated anti-CD4 antibodies,cytofix/cytoperm solution and FITC conjugated anti-IFN-c and IL-4 (BD Pharmingen, USA) before each sam-ple was re-suspended in 500 ll stain buffer. Samples wereanalyzed within 24 h using the Flow-Cytometer and soft-ware described above.

2.7. IL-4 and IFN-c production in culture supernatants

To examine the relationship of CD2 with co-stimulatorysupport provided by macrophages for the release of cyto-kines by T-cells, T-cells (106) from patients and control,

separated using a nylon wool column, were stimulated with4 lg/ml anti-CD2 antibody (BD Pharmingen, USA) eitheralone or in combination with 1 lg/ml anti-CD28 antibody(BD Pharmingen, USA). Control cultures were set up afterpriming with PHA (20 lg/ml). Cells were cultured for 72 hwith whole fixed parasite (10 lg/ml) in the presence orabsence of macrophages obtained from autologousPBMNCs. Cytokine levels (IFN-c and IL-4) were mea-sured in the harvested culture supernatant as previouslydescribed (McKenzie and Zurawski, 1994) by sandwichELISA (Quantikine, R&D Kit, Minneapolis, USA).

2.8. Effect of CD2 on cytokine pattern after treatment with r-

IL-4

Peripheral blood mononuclear cells were primed for 2 hwith Ld antigen (10 lg/ml), treated with 1 lg of humanrecombinant IL-4 (r-IL-4, BD, USA) per ml and culturedin complete RPMI-1640 for 16 h. To confirm the effect ofCD2, simultaneous experiments were performed by co-incubating r-IL-4 treated cells with 4 lg/ml mouse anti-human CD2 MAb (BD, USA). The percentage of CD4+

cells producing either intracellular IFN-c or IL-4 wasdetermined by FACS-Calibur.

2.9. Statistical analysis

Results were analyzed by Mann–Whitney and WilcoxonSigned Rank tests for non-paired and paired samples,respectively. Statistical analysis was performed using SPSSVersion 10.12.

3. Results

3.1. Quantitative T-cell abnormalities due to an inadequate

CD2 expression in VL patients

FACS analysis (Fig. 1a) revealed high levels of CD2antigen on the surface of T-lymphocytes in the controlgroup, which correlated with a high CD4+ cell count inthese subjects. In comparison, T-cells from VL patientsshowed less CD2 expression accompanied with markedlylower CD4+ and CD8+ cell count compared to control.Although both sub-sets showed a reduction in CD2 cellexpression compared to control (P < 0.01), the reductionin CD2 on CD4+ T-cells was more pronounced (Fig. 1b).We next monitored progression of T-cells in cell cycle inthe presence and absence of anti-CD2 (Table 1). A majorityof the T-cells were observed in G0/G1 phase after 16 h ofculture with Ld antigen, which suggest a reduced prolifera-tive state in T-cells during VL infection. There was anincrease in the proportion (%) of T-cells of VL patientsentering S-phase after stimulation with anti-CD2 antibody,which was about 2-fold higher than that of Ld pre-pulsedT-cells in S-phase in absence of anti-CD2 stimulation.Notably, there was substantial and reproducible decreasein the G0/G1 population and a corresponding increase

VLT-cellsCD2 sti. UnSti.

82kDa

VL Control VL Control

Ex Vivo Anti-CD2 Stimulation

0

5

10

15

20

25

30

%C

D4

cells

po

siti

ve t

o P

KC

-al

ph

a/m

icro

litre

Fig. 2. Effect of CD2 activation on cell signaling mechanism in T-cells ofVL patients. Total amount of PKC-a produced by cells stimulated and notstimulated with anti-CD2 antibody evaluated through FACS-Calibur.Immunoblotting of VL T-cells (inset) with PKC-a antibody shows thatanti-CD2 induced significant phosphorylation of a protein migrating at82 kDa.

0

500

1000

1500

2000

2500

3000

VL

Control

VL

Control VL

Control

CD2 CD4 CD8

Cel

ls/M

icro

litre

0

200

400

600

800

1000

1200

VL Control VL Control

CD2+CD4 CD2+CD8

Cel

l/mic

rolit

re

Fig. 1. Immunophenotyping of CD2, CD4 and CD8+ T-cells in VLpatients and control. (a) CD2 down regulation is associated with CD4+ T-cell expression during Visceral Leishmaniasis. Freshly isolated mononu-clear cells (1 · 106) were stained with FITC labeled anti-CD2, FITC or PElabeled anti-CD4 and CD8 antibodies. Flow data were analyzed by usingCell-Quest software on FACS-Calibur. (b) Analysis of CD2+ CD4+ andCD2+ CD8+ cells in T-cell (CD3+) in patients compared to control. Themeans were compared using Mann–Whitney Test. Comparison was madewith the P < 0.05. Abbreviations: VL, Visceral Leishmaniasis.

S. Bimal et al. / Experimental Parasitology 118 (2008) 238–246 241

up to 2.5-fold in T-cells of the G2/M population. We laterexplored specific PKC-a dependant pathway during VLinfection considering its role in growth, differentiationand maturation of T-lymphocytes. There was no significantdifference in the ability of CD4+ cells to produce PKC-a,which was low in both patients and control by FACS anal-

Table 1Inability of T-cells to enter into cell cycle was altered after anti-CD2 stimulat

Categories Stimulants G0/G

VL patients Ex vivo 98.20Ld 97.91Ld + anti-CD2 Ab a 95.36

Control Ex vivo 97.8Ld 97.71Ld + anti-CD2 Ab 96.1

The unstimulated and anti-CD2 antibody stimulated cells from VL patientcontaining trypsin inhibitor and RNase buffer and propidium iodide stain soluusing Modfit software on the FACS-Calibur. Abbreviations: Stim., stimulation

ysis (Fig. 2). PKC production was however higher whenanti-CD2 was added to culture medium containing T-cellsfrom patients (P < 0.05). Western blot results obtainedusing anti-PKC-a antibodies later corroborated the non-significant PKC-a phosphorylation in T-cells of VLpatients without anti-CD2 stimulation (inset in Fig. 2).Rat cerebrum tissue lysate (BD, USA) was run as the acti-vated form of the protein (positive control) in parallel tothe experimental samples (data not shown). Addition ofanti-CD2 induced significant phosphorylation of a proteinmigrating at 82 kDa.

3.2. Increased tendency of T-lymphocytes to converge into

activated blast following stimulation with anti-CD2

Further, complete PBMNCs (5 · 106), pulsed with fixedL. donovani were cultured with anti-CD2 for 16 h and lateranalyzed for the proportion (%) of lymphocytes (LC) andlymphoblasts (LB) in R1 and R2 gated population onFSC vs SSC plot in FACS-Calibur (Fig. 3a). The propor-tion of LB increased after stimulation with anti-CD2 com-pared to that of LC converted into T-LB in L. donovani orPHA stimulated PBMNCs suspensions. In a separateexperiment, on stimulation of PBMNC with anti-CD2, agreater percentage of CD4+ cells became CD69 positive,suggesting that T-cell activation in VL patients isaccounted for by CD2 to some extent (Fig. 3b).

ion

1 S G2M

2 ± 0.48 1.472 ± 0.83 0.327 ± 0.398 ± 0.75 1.178 ± 0.97 0.906 ± 1.336 ± 0.22 2.343 ± 0.23 2.291 ± 0.39

7 ± 1.44 1.07 ± 0.67 1.03 ± 0.870 ± 1.35 0.665 ± 0.94 1.625 ± 2.296 ± 1.02 1.045 ± 0.06 2.89 ± 0.94

s and control were consecutively co-incubated with trypsin, a solutiontion. The stained cells were finally run on a FACS-Calibur and analyzed; Ld, fixed Leishmania donovani parasite; Ab., antibodies.

-20

0

20

40

60

80

100

120

LC LB LC LB LC LB

EX Vivo CD2 Sti. PHA Sti.

Perc

ent o

f Lym

phoc

ytes

and

Ly

mph

obla

st

0

10

20

30

40

50

60

CD69 CD69 CD69

Ex Vivo CD2 Stim. PHA Stim.

%C

D69

pos

itive

CD

4 ce

lls

Gate Gate R1 R2

Fig. 3. Flow cytometry analysis on reversal of T-cell activation pattern in response to L. donovani antigen upon activation of CD2 antigen in VL patients.(a) Conversion of lymphocyte (LC) in lymphoblast (LB): after stimulation of PBMNCs suspension with fixed Ld antigen for 2 h and further cultured infresh complete RPMI-1640 with 4 lg of mouse anti-human CD2 (BD) for 16 h. The frequency of blast cells was analyzed in forward and side scatterhistograms through placement of R1 (on LC) and R2 (on LB). Inset: the forward and side scatter histogram demonstrating the placement of R1 (smalllymphocyte) and R2 (lymphoblast) gates. (b) CD4+ cells activation as CD69 positive cells: cultured cells stimulated or not stimulated with anti-CD2 werewashed and incubated with anti-CD69-PE and anti-CD4-FITC (both from BD, USA) in a single staining step for 15 min in dark and analyzed by flowcytometry.

242 S. Bimal et al. / Experimental Parasitology 118 (2008) 238–246

3.3. Predominance of a well-characterized Th2 cell

associated mechanism during VL infection reverts and moreT-cells show increased sensitivity for IFN-c production after

anti-CD2 stimulation

VL patients and controls were studied in vitro forintracellular cytokine production after stimulation inthe presence or absence of anti-CD2. Analysis was per-formed using R1 for LC and R2 for LB. AnalysingLC in R1 (Table 2 and Fig. 4) it was seen that activation

Table 2Detection of intracellular cytokines produced by lymphocytes of VL patients

Cytokines Perce

Ex vi

IFN- c Total frequency 5.21Contribution of CD4+ cells 46.67

IL-4 Total frequency 43.47Contribution of CD4+ cells 37.74

IL-10 Total frequency 46.63Contribution of CD4+ cells 33.56

Total frequency of cytokine producing lymphocytes and contribution of CD4+

in Fig. 4). Total frequency = (events from upper and lower right of quadrant/tevents from upper and lower right of quadrant) · 100.

of endogenous CD2 antigen by anti-CD2 triggered ahigher frequency of cells to produce IFN-c (3.2-fold)compared to ex vivo. It was 4-fold after stimulation withPHA. The analysis of the total IFN-c production trig-gered due to anti-CD2 showed that only 38% came fromCD4+ T-cells compared to 39.8% accounted by CD4+

cells after PHA stimulation. The R1 gated lymphocyteanalysis further showed that L. donovani infectioninduced a high frequency of IL-4 and IL-10 comparedto IFN-c in the VL subjects. The frequencies of IL-4

and contribution of CD4+ cells (ex vivo and after stimulation)

ntage cells positive ± STDEV

vo Anti-CD2 PHA

± 4.66 16.64 ± 11.66 20.59 ± 11.25± 33.43 37.97 ± 25.82 39.83 ± 23.85

± 18.65 34.46 ± 17.92 48.66 ± 22.88± 5.81 35.64 ± 9.93 35.52 ± 9.97

± 21.71 35.79 ± 19.59 48.69 ± 22.24± 7.24 39.20 ± 9.75 37.93 ± 11.83

T-cells (mean ± SEM) calculated from events of flow data (as representedotal events) · 100. Contribution of CD4+ cells = (events from upper right/

Fig. 4. Increase in frequency of CD4 cells for IFN-c after activation of CD2 antigen in a VL patient: flow diagram of cultured PBMNCs stimulated withor without anti-CD2 or PHA in presence of brefeldin-A and stained with anti-human CD4 PE and anti-human IFN-c FITC of a VL patient showingincrease in frequency of IFN-c producing CD4+ cells (upper right of the quadrant) and IFN-c producing CD4� cells (lower right of the quadrant).

S. Bimal et al. / Experimental Parasitology 118 (2008) 238–246 243

producing cells were low after anti-CD2 stimulation. Fre-quencies of IL-10 producing cells ex vivo and after PHAactivation were similar.

3.4. CD4+ T-cells account for the majority of IFN-cproduced by T-lymphocyte blast after stimulation with anti-

CD2 antibody

In the lymphocyte blast gate R2 (Fig. 5a), there wasdramatic increase in the frequency of IFN-c producing

Fig. 5. T-cell blast shows an increased sensitivity to IFN-c production comparanti-CD2 antibody. (a) Represents histogram obtained on total IFN-c proquadrant). In order to determine the frequency of total activated T-cells or acCD4+ T-cells producing IFN-c, cytokine produced from CD4 (upper right) waData are the mean ± standard deviation for 10 individual patients with VisComparison of all pairs was made with a P value of less than 0.05. *Significant wCD4+ T-cells producing IFN-c amongst the lymphocyte-blast population inlymphocyte was gated on R1.

cells compared to that produced in R1 (Table 2). Thefrequency of LB cells producing IL-4 and IL-10 alsodecreased after anti-CD2 stimulation in VL subjects.Looking further for the contribution of CD4+ LB cells(Fig. 5b), the majority of IFN-c producing LB cellswas shared by CD4+ cells: 54% and 61.6% after stimula-tion, respectively, with anti-CD2 and PHA. However,contribution of CD4+ cells in IL-4 and IL-10 productionbecame low after stimulation with anti-CD2 (data notshown).

ed to IL-4 and IL-10 after the activation of CD2 antigen in VL patients byducing cells in lymphocyte-blast gate R2 (upper right + lower right oftivated CD4 cells producing IFN-c compared to other cells. To determines divided by total cytokine produced by T-cells (upper right + lower right).ceral Leishmaniasis. The means were compared using SPSS version 10.hen compared to Ld stimulated samples. (b) A flow diagram showing % of

a representative VL patient. To analyze total frequency of IFN-c + cells,

0

500

1000

1500

2000

2500

3000

3500

MQ + -- + -- + -- + --

Un sti. Anti-CD2 sti. Anti-CD2+CD28 sti. PHA sti.

IFN

-gam

ma

(pic

ogra

m/m

l. in

cul

ture

su

pern

aten

t)

Fig. 6. CD2 activation in VL patients stimulates T-lymphocytes toproduce IFN-c even in absence of APC. To investigate the requirement forCD2 mediated activation of T-lymphocyte via co-stimulatory supportthrough APC, T-cells (2.5 · 106) were cultured either alone or with2.5 · 106 adherent cells after priming with anti-CD2 antibody either aloneor in combination with anti-CD28 antibody. Control cultures were set upafter priming with PHA. Level of IFN-c was measured by ELISA (asdescribed in Section 2) after 48 h (sti., stimulation; MQ, macrophages; +,in presence of macrophage; �, in absence of macrophages).

244 S. Bimal et al. / Experimental Parasitology 118 (2008) 238–246

3.5. Ability of T-cells to produce IFN-c does not alter in

presence of anti-CD2 even when co-stimulatory support is

removed due to depletion of APC

Although preceding experiments indicated the impor-tance of CD2 antigens in driving a positive shift in theexpansion of IFN-c producing cells, it was unclear whetherthese molecules required APC support in the process. Toaddress this, we examined IFN-c and IL-4 production byELISA in APC supplemented T-cells after 48 h parasiteantigen specific stimulation. In parallel, one experimentalset-up comprised APC depleted T-cells for comparison(Fig. 6). Lack of APCs resulted in lower IFN-c production

0

5

10

15

20

25

30

IFN-y IL4

% C

D4

cell

po

siti

ve t

o c

yto

kin

es

Ex Vivo

CD2

CD2+r-IL4

Fig. 7. CD2 activation in VL patients influences IFN-c responsiveness inT-lymphocytes by down regulating IL-4 production. Cells were pulsedwith L. donovani and were either left unstimulated or stimulated with CD2in presence or absence of recombinant IL-4. Following culture IFN-c andIL-4 pattern in each group was evaluated on FACS-Calibur.

by T-cells in all experimental groups except for unstimu-lated that was equal, however, the decrease was relativelyless after CD2 stimulation compared to anti-CD2 withanti-CD28 and PHA stimulated. In cultures when T-cellswere depleted of APC, anti-CD2 stimulation had little orno impact on IL-4 production (data not shown).

3.6. Effect on IFN-c and IL-4 shift

As shown in Fig. 7, prior treatment of PBMNC withanti-CD2 did not permit CD4+ cells to be properly stimu-lated by recombinant IL-4 and IL-4 production by CD4+

cells, even in presence of r-IL-4, were very low. On theother hand, IFN-c production did not reduce even in pres-ence of r-IL-4 and it increased when stimulated with anti-CD2.

4. Discussion

The role of IFN-c in all forms of leishmaniasis has beenreported to be very important for resistance and cure (Mur-ray, 2001; Murray et al., 2005). Reports of the need for thiscytokine to enable the anti-VL action of pentavalent anti-mony (sb) in experimental infection are also available(Murray and Delph-Etienne, 2000). In this study, wedirectly determined the role of CD2 antigen, a transmem-brane surface adhesion molecule, in promoting prolifera-tive T-cells to transduce efficient signaling for theproduction of IFN-c in VL patients. Direct evidence ofCD2 involvement in immunity to infectious diseases partic-ularly in VL has not been available, although other studieshave demonstrated that CD2–CD58 ligation enhances T-cell antigen recognition (Moingeon et al., 1989; Biereret al., 1989; Van der Merwe, 1989; Bimal et al., 2000,2005; Sasada et al., 2002). This study showed that the num-ber of T-cells (CD3+) expressing CD2 antigen in patientswas lower than that in healthy controls. This reduction inCD2 antigen might affect immunity during VL infectionsince its involvement in T-cell adhesive interaction,enabling an immune response to occur, is well known(Green et al., 2000). The reduction was accompanied witha decline in number of CD4+ cells. Some evidence suggeststhat the activation of Leishmania specific CD8+ cells maycontribute to protective immunity (Titus et al., 1987) andalthough there was a difference in CD8+ cell countsbetween VL patients and healthy donors, the CD8+ cellcount was low in patients in our study. Interestingly, thelevel of CD2 antigen expression in CD4+ and CD8+ cellpopulation was different but the reduction in CD2 antigenon CD4+ cells appeared more marked than that on CD8+

cells. Given that activation of the Th1 population of CD4+

T-cells, which produce IFN-c is instrumental for recovery(Murray, 2001; Murray et al., 2005), it appears that reduc-tion in CD2 antigen on CD4+ cells might interrupt thedevelopment of protective immunity in VL patients. Inanother study it was shown to regulate the positiveselection function of CD8+ T-cells, which is convincing

S. Bimal et al. / Experimental Parasitology 118 (2008) 238–246 245

evidence of its importance in T-cell differentiation function(Teh et al., 1997).

Other studies suggest that CD2 promotes the physicalinteraction of T and NK lineage cells with APCs bearingthe human CD2 ligand, CD58 (Moingeon et al., 1989; Bier-er et al., 1989). As such, to validate the significance of CD2in T-cell function in VL patients, it was important to estab-lish that after stimulation of CD2, T-cells could proliferateand trigger efficient signaling in response to L. donovani.Although not statistically significant, after stimulation ofCD2 on Leishmania sensitized T-cells in VL patients, cellsbehaved differently and progressed more in terms of theproportion of T-cells in G2/M and in S-phase. In contrast,cells from the control group failed to enter into the cellcycle after stimulation with the parasite. As the controlgroup was not exposed, there was no question of antigenpresentation and as such their T-cells had difficulty inentering the cell cycle. There is no data available on roleof CD2 in antigen specific T-cell function and particularlyits direct impact on the cell cycle.

These results are a strong indication that close proximityof CD2 to the region of cell–cell contact after its ligationwith CD58 on APC is crucial to enhance the adhesiveforces to optimize antigen recognition and subsequent T-cell proliferation. Two previous reports, although not anti-gen specific, confirm this hypothesis and we assume thatthis may also be true for VL. Stimulation via CD2 activatesphosphorylation of distinct sites of stathmin (Gouvelloet al., 1998) leading to progression of the cell cycle, whichsuggest that adequate CD2 activation is necessary for T-cells to proliferate in response to L. donovani antigen.Another report links CD2 antigen with T-cell proliferationby demonstrating that it leads to an increased IL-2 produc-tion and receptor expression (Cantrell et al., 1987). Ourdata further suggest that while putative signaling functionof T-cells remains grossly impaired, CD2 stimulationresults in phoshorylation of protein kinase C in T-lympho-cytes which may be an early biochemical event associatedwith T-cell triggering (Monostori et al., 1990). PKC isknown to be a key regulator or mediator of T-cell activa-tion and is capable of activating T-cell for cytokine produc-tion (Bhattacharya et al., 2001). Stathmin is a target for thecell cycle and in context with our results; it is possible thatthe activation of diverse protein kinase systems such asPKC by CD2 promoted the stathmin pathway that trig-gered cells to proliferate in response to Leishmania (Bhat-tacharya et al., 2001).

At this point, given the fact that CD2 was a key candi-date in driving a pronounced change in cell cycle behaviorand signal transduction ability of T-cells, we attempted torelate this with the potential of CD2 activation in patientsto induce a protective response in CD4+ cells. That thestimulation of CD2 present in patients by anti-CD2 anti-body induced a marked increase in lymphoblast cells illus-trates how a lack of activation of CD2 would haveimpaired the L. donovani recognition process of T-cells.The other significant finding was that the activation of

CD2 amplified the potential of CD4+ cells, which on aver-age became more CD69 positive. The present study showedthat both Th1 and Th2 cell response was triggered duringL. donovani infection, although the relative over expressionof Th2 cell associated mechanisms overshadowed the pro-tective Th1 response (Karp et al., 1993; Sunder et al.,1997). The study further showed that after triggering ofCD2, the percentage of IFN-c-positive CD4+ cells was ele-vated, but the increase in lymphoblast population of CD4+

T-cells was significantly greater. Earlier, when such cellswere not activated, IL-4 and IL-10 producing T-helper cellswere much more frequent and there was no instance inwhich a protective mechanism was apparent in these VLpatients. The significance of this finding is that CD2 activa-tion has a strong impact on CD4+ cells, which are activatedand accounts for the majority of the IFN-c producing cellsduring VL infection. It was also shown that T-lymphocytesenhanced their activation for the secretion of IFN-c in theabsence of APC and that a stimulation of CD2 wasrequired for this effect (Meinl et al., 2000).

Previous studies illustrate that IL-12 production by NKcells and macrophages is critical in the development of Th1cells in response to infection with Leishmania (Murray andDelph-Etienne, 2000). We also showed that CD2 mediatedreconstitution of a protective IFN-c dominant immuneresponse is triggered through an IL-4 down regulatingmechanism in VL patients. Over expression of an IL-4and IL-10-biased CD4 response during VL, antagonizesprotective IFN-c response and leads to inhibition ofimmune control in vivo, resulting in disseminated formsof the disease. Hence, evidence for the role of CD2 antigenin immunity to L. donovani infection shown in this study isworth pursuing.

Acknowledgments

We are indebted to Dr. Philippe Desjeux, One WorldHealth, USA; Dr. Jenifer Blackwell, UK and Dr. ShyamSunder, BHU, Varanasi, India for valuable suggestionsduring the course of this investigation. Technical servicesrendered by Mr. Arvind Prasad, Technical Assistant,Immunology Division, RMRIMS, India is highly appreci-ated and acknowledged.

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