Table 3.

Human studies on ENL investigating T-cell biology.

Reference; study site(s)	Study population	Timing of sampling	MDT status	ENL treatment status	Type of samples	Measures	Findings
Lim et al. (84); USA and Korea (mixed ethnic background)	7 LL ENL, 20 active LL, 9 inactive LL, 4 BB, 3 indeterminate leprosy	ND	All patients treated with Dapsone or Clofazimine or Rifampicin for varying durations	5 had received various doses of steroids and 3 were treated with steroids at the time of the study	Blood	T lymphocyte numbers by the rosette assay	ENL showed T-lymphocyte numbers significantly higher than LL
							LL had lower T-lymphocyte numbers than HC
Anders et al. (119); Papua New Guinea	31 leprosy: 13 BL/LL with amyloidosis (11/13 frequent ENL), 9 BL/LL ENL without amyloidosis, 9 BL/LL with few or no ENL episodes	ND	Approximately half patients on clofazimine and other half on dapsone	2 ENL at testing: 1 steroids and 1 stibophen	Blood	Lympohocyte transformation tests	Patients with a history of frequent ENL had greater cell-mediated responses to PHA than patients without ENL
Izumi et al. (70); Japan β	12 ENL, 49 active lepromatous, 24 inactive lepromatous, 7 borderline, 6 tuberculoid, 9 HC	ND	ND	ND	PBMC	Percentage and number of Tμ (T cells with Fc receptor for IgG) and Tγ (T cells with Fc receptor for IgM)	No significant differences between different clinical groups
Bach et al. (86); France (multiple ethnic groups)	9 BL/LL with no recent history of ENL, 9 BL/LL suffered from ENL less than 2 months prior to the investigation, 13 BT/TT, HC	ND	Some untreated and others on MDT	Certain ENL on antireactional treatment	Blood	T cell subsets; Proliferative responses to mitogens	Increased %age of helper T cells in ENL
							Decreased %age of suppressor T cells in ENL
							Elevated proliferative responses to mitogens in ENL
						Con A-induced suppressive activity	Most ENL decrease of suppressive index, whereas none of the LL or TT patients had a diminished suppressive activity
Dubey et al. (120); India	41 untreated cases of leprosy, 64 TT and LL taking antileprosy treatment, reactional (8 ENL and 10T1Rs), 11/41 follow-up from untreated leprosy patients	ND	64 cases on antileprosy treatment	Untreated cases of ENL?	Blood	Lymphocytic culture: percentage of Blast transformation	Blast percentage in ENL slightly higher than T1R
Mshana et al. (90); Ethiopia	21 BL/LL, 10 BT, 5 ENL	ND	All patients received MDT but unclear whether sampled prior to MDT	No patient on thalidomide	Blood	Lymphoproliferative responses to PPD or PHA T-cell subsets	Higher responses to PPD or PHA in ENL Decreased number of suppressor cells prior to ENL, which increased with clinical recovery from ENL
Mshana et al. (88); Ethiopia	69 leprosy patients: 26 ENL, 13 HC	Untreated samples	Untreated samples	Untreated samples	Blood	T lymphocyte subpopulations; lymphoproliferation using M. leprae, PHA and PPD	ENL patients had decrease in suppressor cells and an increase of CD4+/CD8+ ratio compared to LL ENL had higher responses to both PHA and PPD BL/LL patients with or without ENL lower proliferative responses to M. leprae than BT patients and HC
Wallach et al. (87); France (samples from multiple ethnic groups)	9 recent ENL, 6 bacteriologically positive patients of which 1 ENL more than 5 years ago, 9 treated leprosy patients of which 3 had ENL	Described in detail each patient duration of disease	All treatment described in detail	Some on antireactional treatment	Blood	T cell subsets; Lymphocyte transformation tests: proliferative responses to mitogens	ENL patients have elevated Helper/Suppressor ratio
							Mean proliferative responses elevated in ENL
Bach et al. (121); France	8 treated lepromatous without recent ENL with BI < 1 +, 6 lepromatous with BI > 2+ (untreated or suffering a relapse, without recent ENL reaction), 12 lepromatous who underwent at least one ENL episode, 13 tuberculoid, 41 HC	ND	ND	ND	PBMC	T-cell subsets; Proliferative response to M. leprae and PPD of isolated T-cell subsets	ENL decreased CD8+ T cell percentages and increased CD4+/CD8+ ratios T-cell subset percentages returned to normal either when the bacterial load was reduced by treatment or when the ENL reaction resolved ENL episodes associated with improvement of T-cell unresponsiveness to various antigens or mitogens
Modlin et al. (97); USA	15 non-reactional leprosy BT/BB/BL/LL, 17 reactional (6 T1R, 9 ENL, 2 Lucio’s reaction)	ND	Results did not differ between treated and untreated subjects	3 ENL had no therapy	Skin	T lymphocyte subsets	The helper/suppressor ratio in ENL was significantly higher than in non-reactional lepromatous disease
Modlin et al. (98); USA	14 leprosy patients (4 tuberculoid, 2 borderline in T1R, 1 BL, 7 lepromatous of which 5 ENL), 8 HC	ND	6 treated patients	ND	Skin	T lymphocyte subsets	ENL lesions showed 2:1 predominance of helper cells whereas in the lesions without ENL the helper: suppressor ratio was 1:1 smaller
Sasiain et al. (122); Argentina	16 ENL, 12 HC	First blood sample ND; 9 ENL 20-30 days after stopping thalidomide	All patients on MDT	Thalidomide in patients with ENL	PBMC	ConA-induced suppressor response	Suppressor T-cell function was reduced during ENL and after ENL than HC
Narayanan et al. (89); India ε	7 LL ENL, 6 BT T1R, 5BL T1R, 18 BT-LL	ND	ND	ND	Skin	T cell phenotypes	Lesions of ENL showed increase in T cells with a predominance of the helper/inducer subset; CD4+/CD8+ ratio was higher in ENL and T1R than non-reactional lesions
Rea et al. (96); USA ε	19 ENL, 24 LL non-reactional with treatment, 12 LL non-reactional no treatment, 18 LL with long-term treatment, 4 LL with Lucio’s, 13 BL, 13 T1R, 18 Tuberculoid, 13 Tuberculoid with long-term treatment	ND	Some patients on MDT	ENL before receiving thalidomide	PBMC	T cell subsets	Active LL patients have lymphopenia, a proportionate reduction in the numbers of each of the three T cell subsets
							Insignificant changes in T cell subsets expressed as percentages and in the helper: suppressor ratio
Laal et al. (94); India ε	15 ENL, 13 LL	During active ENL and 1 week to 4 months after stopping treatment	On MDT	First sample before initiation of antireactional treatment	Blood	Leukocyte migration inhibition test	ENL significant inhibition of antigen-induced leukocyte migration
						Lymphoproliferation	Lymphoproliferation enhanced during the acute phase of ENL
				Second sample 1 week to 4 months after stopping treatment		Suppressor cell activity; T cell subsets	Enhanced antigen-stimulated suppression of mitogen responses in ENL
							Leukocyte migration inhibition, lymphoproliferation, and suppressor cell activity were reduced in post-ENL to the unresponsive state seen in stable LL
							Lower CD4+/CD8+ ratio in ENL compared to LL
Modlin et al. (99); USA	12 ENL and 10 non-reactional leprosy; 19 ENL blood samples	ND	ENL biopsies: 8/12 treated with dapsone; ENL blood: 15/19 treated	Some ENL were treated	Blood	T lymphocyte subsets	ENL tissue more cells of the helper-inducer phenotype and fewer of the suppressor-cytotoxic phenotype, as compared with non-reactional LL
					Skin		No correlation between tissue and blood helper-suppressor ratios
Wallach et al. (91); France	ND	ND	ND	ND	Blood	T cell helper-suppressor (HS) ratio	HS ratio higher in ENL lesions and blood than non-ENL leprosy controls
					Skin
Modlin et al. (100); USA	Biopsies: 25 ENL, 23 tuberculoid, 23 non-reactional lepromatous;	ND	Some patients received treatment	Some patients on treatment?	Blood	Skin: number of T cells, T cell subsets; Blood: lepromin-induced suppression of the Con A stimulation	Increases in both CD4+/CD8+ ratio and the number of IL2-positive cells in ENL Suppressor activity decreased significantly in ENL Suppressor activity returned to normal after ENL subsided
	Blood: 18 ENL
					Skin
Rao and Rao (123); India ε	44 ENL, 39 BL/LL, 22 post-ENL	ENL patients before starting ENL treatment, post-ENL after patient had not taken anti-inflammatory/steroids for at least 3 and 7 days	From 39 non-reactional cases: 20 untreated and 19 with dapsone for less than a year	Before starting treatment for ENL with steroids or anti-inflammatory drugs, post-ENL: ensuring that the patient had not taken anti-inflammatory drugs or steroids for at least 3 and 7 days, respectively	Blood	Sub-population of T cells with receptors for Fc portion of IgG (Tr) and Fc portion of IgM (Tμ)	Tμ/Tr ratio higher in ENL than lepromatous and post-ENL patients
Rao and Rao (85); India	77 leprosy: 44 ENL	ENL: before starting anti-ENL treatment, post-ENL: After patient had noe taken anti-inflammatory drugs or steroids for at least 3 and 7 days	19 patients treated with dapsone for less than 1 year	Before starting treatment for ENL with anti-inflammatory drugs or steroids	Blood	Leykocyte migration inhibition test (LMIT)	No significant difference in mean migratory index to PHA, PPD, sonicate M. leprae
						Enumeration of early and total T lymphocytes	Whole M. leprae increased response in ENL compared to LL Lower migratory indices to whole M. leprae in post-ENL than LL
							%age of early T lymphocytes increased in ENL compared to LL
							%age of early T lymphocytes remained high in post-ENL compared to LL
							Cell-mediated immune responses enhanced during ENL and return to LL levels once the episode is over
Shen et al. (101); USA	10 ENL, 8TT/BT, 10 BL/LL, 10 T1R	ND	ND	ND	Skin	CD3, CD4, CD8 and Ta1 (memory) positive cells	CD3, CD4 and CD8 showed percentages of positive cells in lesions similar between patient groups
					PBMC		No significant difference in%age of memory T-cells in ENL compared to LL
Bottasso et al. (124); Argentina	8 LL/ENL, 17 LL, 9 TT, 11 HC	ND	Patients on MDT	Patients with ENL were not on thalidomide treatment but unknown whether they were on steroids	Blood	T-Lymphocytes count absolute and relative; Lymphocyte functional assay: capacity of rosetta formation	Active LL showed a decrease in T-lymhocytes
							ENL showed a restoration of the levels of T-lymphocytes
Rasheed et al. (125); Zambia and Pakistan	167 leprosy of which 21 LL/ENL, 12 BL/T1R, 24 BT/T1R, 46 endemic HC	ND	ND	ND	Serum Lymphocytes	Lymphocytotoxic activity	Lymphocytotoxic activity scores were significantly raised in patients with reactions
Sasiain et al. (126); Argentina	53 leprosy patients TT/BT/BB/BL/LL and 9 LL/ENL, 23 HC	ND	Received MDT	Thalidomide for ENL	PBMC	Proportion of CD8+ cells	Proportion of CD8+ cells was low in LL patients and tended to normalize during ENL episodes
						M. leprae-induced suppression of T-cell proliferation; Induction of IL-2R by culture with M. leprae
						PHA- and ConA-induced proliferation
Bhoopat et al. (127); Thailand ε	57 ENL (19 acute/38 chronic), 61 active LL, 33 cured leprosy	26 BL/35 LL newly diagnosed and untreated	ND	If corticosteroid and/or thalidomide was initiated before or during the study, precise timing of medication was recorded with respect to the time of collection of laboratory specimens	Blisters induced over a representative skin lesion	T cell subsets in situ	The lesions of chronic ENL showed a decreased number of CD8+ cells and increased helper/suppressor ratio compared to those in acute ENL and non-reactional leprosy; Systemic administration of corticosteroids caused a reduction in the CD4+ cell population but did not change CD8+ cell population
Rea and Modlin (102); USA δ	ND	ND	ND	ND	Skin	T-cell phenotypes: CD4+ versus CD8+ cells, γ/δ and α/β receptor-bearing lymphocytes, T-memory and T-naïve cells	ENL lesions predominance of CD4+ cells similar to those in tuberculoid (TT/BT?) and T1R
							LL patients showed an excess of CD8+cells
Tyagi et al. (53); India	4 TT/BT, 5 BL/LL, 4 ENL	ND	ND	ND	Blood	Effect of isolated circulating ICs from BL/LL or ENL patients to lymphocyte transformation test on T cells of HC	PEG precipitates isolated from BL/LL or ENL subjects had a significant suppressive effect on lymphocyte proliferation in HC
Foss et al. (128); Brazil δ	28 lepromatous: 11 ENL, 23 tuberculoid, 19 HC	ND	lepromatous patients 86% treated with dapsone	11 ENL at time of blood collection no immunosuppressive drug	Blood	T lymphocyte response to concanavalin A	Marked reduction on concanavalin A-induced lymphoproliferation in patients with ENL
Santos et al. (129); Brazil ε	59 LL/BL, 10 ENL, 4 T1R, 4 post-reactional	ND	On MDT	No specific treatment for reactions before blood collection	PBMC	Lymphocyte proliferation after ConA and M. leprae	T1R showed greater lymphocyte proliferation compared to all other groups
de la Barrera et al. (130); Argentina	7 TT/BT, 20 BL/LL of which 3 ENL	ND	All patients on MDT	ND	PBMC	T-cell cytotoxic activity induced by M. leprae and M.tb heat shock protein (HSP)	M. leprae hsp65 induced cytotoxic responses only in those MB patients undergoing ENL
Vieira et al. (131); Brazil δ, ε	95 MB leprosy (30LL/65BL) of which 51 ENL	At leprosy diagnosis and at onset of reactional episode	Time of MDT for each ENL	Sample before thalidomide and steroids?	PBMC	Lymphocyte transformation test (LTT)	Some patients showed lymphoproliferative response during ENL
Mahaisavariya et al. (103); Thailand	17 non-reactional, 8 T1R, 12 ENL	Biopsy at the time of diagnosis and not the time of reaction	ND	ND	Skin	T-lymphocyte subsets	%age of CD8 infiltration reduced in ENL compared with non-reactional lepromatous
							The CD4+/CD8+ ratio of ENL statistically significant higher than from the non-reactional lepromatous group
Tadesse et al. (132); Ethiopia δ	33 leprosy: 14 BT, 11 BT T1R, 8 ENL, 11 HC	ND	Certain leprosy patients were treated on MDT	All ENL treated with steroids	PBMC	Lymphocyte blast transformation	Thalidomide treatment did not alter the lymphoproliferative response to the mycobacterial antigens during ENL
Mohanty et al. (133); India	21 BL/LL ENL, 38 TT/BT/BL/LL, 29 BT/BL T1R, 19 HC	ND	ND	ND	PBMC	Immune responses against Stress proteins of M. leprae (lymphoproliferation)	ENL: no significant role of stress proteins except a heightened lymphoproliferative response to the 28 kDa antigen
					Serum
Villahermosa et al. (134); Philippines δ, ε	22 ENL	Before thalidomide and at study weeks 3 and 7 during thalidomide	MDT continued during the study	Samples untreated for antireactional drugs and during thalidomide treatment	Blood	Lymphocyte proliferation assays (LPA) to phytohemagglutinin and concanavalin A	Low LPA values pre-thalidomide in both PBMC and whole blood
					PMBC
Attia et al. (107); Egypt	38 leprosy: 6 ENL, 38 HC	Untreated samples	Untreated samples	Untreated samples; excluded patients on immunosuppressive drugs	Blood	Frequency of circulating Tregs; FoxP3 expression	Significantly lower frequency of Tregs but higher FoxP3 expression in ENL
Massone et al. (116); Brazil ε	20 leprosy: 3 ENL	Biopsies at the time of diagnosis	10, 12 and 13 months after beginning of MDT for LL	Untreated for antireactional treatment	Skin	Presence, frequency and distribution of Tregs	No statistical difference in FoxP3 expression between TT, BT, BL, and LL
							Significant increase in FoxP3 expression in T1R compared to ENL
Rada et al. (135); Venezuela ε	? ENL81 LL, 41 BL, 41 BB, 3% BT	ND	ND	ND	Blood	Cell-mediated immunological tests to mycobacterial proteins	T-lymphocyte proliferative response in reactional and non-reactional patients was negative
Saini et al. (136); India δ	21 MB: 16 ENL, 5 T1R	ENL blood during reaction and at 0.5 and 1 year after the onset of reaction	Duration of MDT described	ENL patients received steroids	PBMC	Lymphoproliferation of PBMC stimulated with M. leprae, recombinant Lsr2 and 6 synthetic peptides spanning the Lsr2	All patients with active ENL showed lymphoproliferation in response to peptides A and F
Abdallah et al. (109); Egypt δ	43 leprosy: 6 ENL, 40 HC	Untreated patients	Untreated samples	Untreated	Blood	Circulating Tregs	Tregs/Teffs lowest in ENL
Attia et al. (110); Egypt δ	43 leprosy: 6 ENL, 40 HC	Untreated patients	Untreated samples	Untreated	Blood	CD4(+) CD25(high)Foxp3 (+) regulatory cells	CD4(+)CD25(high)FoxP3(+) Treg levels lowest in ENL
							Treg/Teffs lowest in ENL
Hussain et al. (92); India	50 leprosy (28 without reactions, 11 T1R, 11 ENL), 50 HC, 50 pulmonary TB (25 HIV-TB co-infected and 25 without HIV infection), 50 HIV-positive	ND	Reactional episodes following antileprosy treatment	ND	Blood	CD3+, CD4+, CD8+ and CD4+/CD8+ ratio with flow cytometry	CD4+ counts raised during ENL compared to MB patients whereas CD8+ counts lower The CD4+/CD8+ ratio doubled during reactional episodes of T1R and ENL
Parente et al. (137); Brazil	2 ENL, 103 leprosy TT/BT/BB/BL/LL 9 indeterminate, 8 T1R	2 ENL: 12 and 10 months after initiation of MDT	2 ENL after initiation of MDT	ND	Skin	Frequency and distribution of regulatory T cells	No significant differences in ENL
Saini et al. (113); India δ	66 leprosy: 15 T1Rs, 15 ENL, 36 BT/LL	Newly diagnosed leprosy patients prior to institution of antireaction therapy	Freshly diagnosed patients: untreated subjects	Newly diagnosed leprosy patients prior to institution of antireaction therapy	PBMC	MLSA stimulated and unstimulated PBMC: gene expression with PCR array for 84 genes; T cell phenotypes	Increase in FOXP3 gene expression in ENL
							Th17 cells with intracellular IL-17A, F are increased in ENL and CD4+IL-21+ cells are higher in ENL
							Significant upregulation of CD4+CCR6+ cells in ENL
							Tregs decreased in ENL

α, also in Table 1; β, also in Table 2; δ, also in Table 4; ε, also in Table 5.

BB, mid-borderline leprosy; BL, borderline lepromatous leprosy; BT, borderline tuberculoid leprosy; ENL, erythema nodosum leprosum; HC, healthy controls; HS, helper-suppressor; HSP, heat shock protein; ICs, immune complexes; LL, lepromatous leprosy polar; LTT, lymphocyte transformation test; LPA, lymphocyte proliferation assay; MB, multibacillary; ND, not described; PEG, polyethylene glycol; PHA, purified phhytohaemagglutinin; PPD, RT23 tuberculin-purified protein derivative; SLE, systemic lupus erythematosus; TB, tuberculosis; TT, tuberculoid leprosy polar.