A human immune data-informed vaccine concept elicits strong and broad T-cell specificities associated with HIV-1 control in mice and macaques

Background: None of the HIV T-cell vaccine candidates that have reached advanced clinical testing have been able to induce protective T cell immunity. A major reason for these failures may have been suboptimal T cell immunogen designs. Methods: To overcome this problem, we used a novel immunogen design approach that is based on functional T cell response data from more than 1,000 HIV-1 clade B and C infected individuals and which aims to direct the T cell response to the most vulnerable sites of HIV-1. Results: Our approach identified 16 regions in Gag, Pol, Vif and Nef that were relatively conserved and predominantly targeted by individuals with reduced viral loads. These regions formed the basis of the HIVACAT T-cell Immunogen (HTI) sequence which is 529 amino acids in length, includes more than 50 optimally defined CD4+ and CD8+ T-cell epitopes restricted by a wide range of HLA class I and II molecules and covers viral sites where mutations led to a dramatic reduction in viral replicative fitness. In both, C57BL/6 mice and Indian rhesus macaques immunized with an HTI-expressing DNA plasmid (DNA.HTI) induced broad and balanced T-cell responses to several segments within Gag, Pol, and Vif. DNA.HTI induced robust CD4+ and CD8+ T cell responses that were increased by a booster vaccination using modified virus Ankara (MVA.HTI), expanding the DNA.HTI induced response to up to 3.2% IFN-γ T-cells in macaques. HTI-specific T cells showed a central and effector memory phenotype with a significant fraction of the IFN-γ+ CD8+ T cells being Granzyme B+ and able to degranulate (CD107a+). Conclusions: These data demonstrate the immunogenicity of a novel HIV-1 T cell vaccine concept that induced broadly balanced responses to vulnerable sites of HIV-1 while avoiding the induction of responses to potential decoy targets that may divert effective T-cell responses towards variable and less protective viral determinants.

Avaliação do uso de extrato de timo (timulina) em pacientes com neoplasia maligna submetidos ao tratamento cirúrgico

OBJETIVO: Avaliar a ação da timulina em pacientes com neoplasia maligna, submetidos ao tratamento, com e sem quimioterapia e radioterapia complementar (QT/RT). MÉTODO: Estudo retrospectivo realizado em 50 pacientes, analisando as variações das taxas de leucócitos, linfócitos totais e da relação de linfócitos CD4/CD8 após imunoestimulação com timulina. RESULTADOS: No grupo submetido à QT/RT ocorreu um aumento do número de leucócitos após seis meses em 43,33% dos casos, e em 83,33% após 12 meses. Com relação aos linfócitos totais, após seis meses, 63,33% apresentaram níveis maiores, e depois de 12 meses isto ocorreu em casos 90% dos casos. A relação de linfócitos CD4/CD8 mostrou um aumento em 66,66%, e em 90% depois após 6 e 12 meses respectivamente. A análise estatística se mostrou significante com o teste de ANOVA one way. No grupo não submetido à QT/RT a elevação dos níveis de leucócitos ocorreu em 85% dos pacientes aos seis meses e em 90% aos 12 meses. As taxas de linfócitos se elevaram em 60% dos casos em seis meses e em 85% após 12 meses. A relação CD4/CD8 se tornou maior tanto aos seis como aos 12 meses em 65%. A análise estatística mostrou relevância com o teste "t" de Student e o de ANOVA one way. Não houve necessidade de interrupção dos ciclos de QT/RT e nenhum paciente referiu intolerância à timulina. CONCLUSÃO: O uso da timulina foi capaz de restaurar a resposta imune, reduzir os danos imunossupressores e colaterais induzidos pela terapia antineoplásica e não apresentou efeitos colaterais.

Quantificação das populações e subpopulações de linfócitos em gestantes com pré-eclâmpsia

OBJETIVO: analisar a relação do sistema imunológico com a pré-eclâmpsia, comparando-se populações e subpopulações de linfócitos em gestantes saudáveis e com pré-eclâmpsia. MÉTODOS: foram estudadas 40 gestantes, sendo 20 saudáveis e 20 com doença hipertensiva específica da gestação. Amostras de sangue periférico foram obtidas de todas as gestantes e realizou-se quantificação de populações e subpopulações de linfócitos (CD4+, CD8+, relação CD4+/CD8+, CD3+ e CD19+). A análise estatística foi realizada pelo teste de Mann-Whitney. RESULTADOS: os resultados encontrados foram: contagem de linfócitos menores nas pré-eclâmpticas (grupo de estudo 2295,10±1328,16, grupo controle 3892,80±1430,85, p<0,05), assim como de CD4+ (grupo de estudo 1188,80±625,61, grupo controle 1742,25±628,40, p<0,05), CD8+ (grupo de estudo 774,00±371,31, grupo controle 1175,70±517,72, p<0,05), CD3+ (grupo de estudo 1958,65±983,78, grupo controle 2916,95±1117,88, p<0,05). Os demais testes não apresentaram diferenças estatisticamente significantes nos dois grupos. CONCLUSÕES: conclui-se que há diminuição no número de linfócitos, CD4+, CD8+ e CD3+ na pré-eclâmpsia.

Diversidade genética do vírus da imunodeficiência humana tipo 1 (HIV-1) em mulheres infectadas de uma cidade do nordeste do Brasil

OBJETIVO: descrever a diversidade genética dos isolados de HIV-1 de mulheres soropositivas acompanhadas em um centro de referência. MÉTODOS: estudo transversal, no qual foram incluídas 96 mulheres com dois testes sorológicos ELISA e um teste confirmatório Western Blot. Das amostras de sangue periférico, foram determinadas a carga viral pelo kit b-DNA e a contagem de linfócitos T CD4 e T CD8 pela citometria de fluxo excalibur. A extração e purificação do DNA pró-viral foi realizada pela reação em cadeia da polimerase (PCR), utilizando o kit QIAamp Blood (Qiagen Inc., Chatsworth, CA, USA). O sequenciamento da região pol foi realizado em 52 isolados com o (3100 Genetic Analyzer, Applied Biosystems Inc., Foster City, CA) e a genotipagem foi investigada pela ferramenta Rega (Rega Subtyping Tool). O padrão de resistência aos antirretrovirais (ARV) foi inferido pelo algoritmo do banco de dados Stanford HIV Resistance. Os estágios clínicos das participantes foram definidos como A, B ou C segundo os critérios do Center for Diseases Control (CDC). Para a análise estatística dos dados, foram utilizados os testes do χ2 para as variáveis categóricas e o teste t de Student para as variáveis numéricas. RESULTADOS: a média de idade da amostra, o tempo médio de doença e de tratamento foram: 33,7; 3,8 e 2,5 anos, respectivamente. A média da carga viral foi log10 2,3 cópias/mL; a dos linfócitos T CD4 e T CD8 foi 494,9 células/µL e 1126,4 células/µL. Sobre o estágio clínico, 30 mulheres estavam no estádio A, 47 no B e 19 no C. O sequenciamento dos 52 isolados encontrou 33 do subtipo B, quatro do F, um do C e 14 do recombinante BF. A análise da resistência aos ARV mostrou 39 (75,0%) isolados susceptíveis, 13 (25,0%) resistentes aos inibidores da transcriptase reversa (INTR) e três (5,7%) aos inibidores da protease (IP). CONCLUSÕES: Houve grande diversidade do HIV-1 e elevado percentual de isolados resistentes aos ARV na amostra estudada.

Thymic atrophy in cattle poisoned with Solanum glaucophyllum

Solanum glaucophyllum (Sg) [= S. malacoxylon] is a calcinogenic plant inducing "Enzootic Calcinosis" in cattle. The 1,25-dihydroxyvitamin D3, its main toxic principle, regulates bone and calcium metabolism and also exerts immunomodulatory effects. Thymocyte precursors from bone marrow-derived progenitor cells differentiate into mature T-cells. Differentiation of most T lymphocytes is characterized not only by the variable expression of CD4/CD8 receptor molecules and increased surface density of the T cell antigen receptor, but also by changes in the glycosylation pattern of cell surface glycolipids or glycoproteins. Thymocytes exert a feedback influence on thymic non-lymphoid cells. Sg-induced modifications on cattle thymus T-lymphocytes and on non-lymphoid cells were analysed. Heifers were divided into 5 groups (control, intoxicated with Sg during 15, 30 or 60 days, and probably recovered group). Histochemical, immunohistochemical, lectinhistochemical and morphometric techniques were used to characterize different cell populations of the experimental heifers. Sg-poisoned heifers showed a progressive cortical atrophy that was characterized using the peanut agglutinin (PNA) lectin that recognizes immature thymocytes. These animals also increased the amount of non-lymphoid cells per unit area detected with the Picrosirius technique, WGA and DBA lectins, and pancytokeratin and S-100 antibodies. The thymus atrophy found in intoxicated animals resembled that of the physiological aging process. A reversal effect on these changes was observed after suppression of the intoxication. These findings suggest that Sg-intoxication induces either directly, through the 1,25-dihydroxyvitamin D3 itself, or indirectly through the hypercalcemia, the observed alteration of the thymus.

Comportamento de células do sistema imune frente ao desafio com Salmonella Enteritidis em aves tratadas e não tratadas com ácidos orgânicos

A Salmonelose é uma importante zoonose, considerada a principal causa de infecções bacterianas, sendo associada ao consumo de produtos avícolas. Como alternativa de controle, ácidos orgânicos têm sido amplamente usados. No entanto, pouco se conhece sobre o estado imunológico de aves de produção, e uma avaliação deste status é necessária para proteger frente a enfermidades e para garantir à aplicação segura de agentes terapêuticos ou imunização profilática. Este trabalho teve como objetivo verificar o comportamento do sistema imunológico das aves previamente infectadas com Salmonella Enteritidis (SE) tratadas com um composto de ácidos orgânicos em diferentes concentrações administrado via água e ração comparando com as aves infectadas e não tratadas. Foram inoculados 120 frangos de corte com 1mL de SE, via oral, na concentração de 1,0 x 108 UFC/mL, no 1º e 2º dia de idade, divididos em seis tratamentos com duas repetições, utilizando 200, 400, 500 e 1000ppm do ácido orgânico. Aos 35 dias de vida das aves, foram coletados, de todos os grupos, alíquotas de sangue de 3mL em tubo contendo EDTA para a avaliação das células imunes através de citometria de fluxo. Foram analisadas as porcentagens circulantes de células CD4+, CD8β+, MHC I+, MHC II+, TCRVβ1+, TCRVβ2+ e CD28+. Para análise microbiológica foram coletadas tonsilas cecais destas aves. Observou-se com esse estudo que os ácidos orgânicos nas dosagens 1000ppm na água e 500ppm na ração durante, dois e sete dias respectivamente antes do abate, foram eficazes na redução da infecção por SE em frangos de corte, comprovadas pelo método microbiológico e demonstradas através do comportamento das células do sistema imune. No presente estudo as aves infectadas apresentaram uma proporção menor de células T auxiliares circulantes quando comparadas às aves infectadas, mas tratadas com o AO ou com o grupo não infectado. A mesma tendência pode ser observada para as células CD28+, TCRVβ1+ e MHC IIbright+, e, com menor resolução, para CD8β+.

Uso de probiótico sobre a ativação de células T e controle de Salmonella Minnesota em frangos de corte

Para avaliar o efeito do probiótico sobre a resposta imunológica de frangos de corte desafiados com Salmonella Minnesota (SM), 60 frangos foram divididos em três grupos: CN- (controle negativo) aves que não foram inoculadas com SM, CP- (controle positivo) aves inoculadas com SM e Probiótico- aves suplementadas na ração com probiótico composto de Lactobacillus acidophilus, L. plantarium, L. rhamnosus, L. bulgaricus, Enterococcus faecium, Streptococcus thermophilus e Bifidobacterium bifidum e desafiadas com SM. Aos 14 dias foi realizada a inoculação com SM e aos 7 e 35 dias foram quantificadas células caliciformes, CD4+ e CD8+ na mucosa intestinal do íleo e ceco. Aves suplementadas com probióticos aos 7 dias de idade apresentaram aumento significativo (P≤0,05) de células caliciformes e CD4+ no íleo e de células CD8+ no ceco. Aos 35 dias houve aumento significativo (P≤0,05) das células CD8+ nas aves inoculadas do CN e Probiótico. A utilização de probióticos proporcionou redução significativa (P≤0,05) da contagem de Salmonella sp.

Induction of immune response in broiler chickens immunized with recombinant FliC and challenged by Salmonella Typhimurium

The study examined (1) the immune response in broiler chickens after oral immunization with recombinant flagellin (rFliC) from Salmonella Typhimurium conjugated with sodium alginate microparticles, and the immune response enhancement in association with recombinant cholera toxin B subunit protein (rCTB) and pool of Lactobacillus spp. (PL). The immune responses were evaluated by dosage of IgY serum and IgA from intestinal fluid and immunostaining of CD8+ T lymphocytes in the cecum. The immunized animals were challenged with Salmonella Typhimurium (ST) 21 days after treatment. In all immunized groups, a significant increase (p<0.05) was observed in IgA levels (μg/mL), especially three weeks after immunization. The serum IgY levels (μg/mL) were little affected by the treatments and differed significantly among groups only in the second post-immunization week (p<0.05). After the challenge, the number of CD8+ T cells differed significantly between the treatments and negative control. Retrieval of Salmonella Typhimurium was not detected at 48 hours after the challenge in T2 (rFliC+rCTb), T3 (rFliC+PL) and T4 (rFliC+rCTB PL). The rFliC administered orally with or without rCTB and Lactobacillus spp. produces significant induction of humoral immune response, and the immunized chickens were more effective in eliminating Salmonella after challenge.

Cellular immune response of Curraleiro Pé-duro and Nellore calves following Mycobacterium bovis-BCG vaccination

The present study aimed to assess the CD4, CD8 and γδ blood levels for Curraleiro Pé-duro, as well as the specific IFN-γ response after BCG vaccination using flow cytometry. The specific immune response against BCG was also evaluated by tuberculin skin test, performed before and 45 days after the vaccination. For comparison purposes, the same parameters were investigated on Nellore calves, an exotic bovine with resistance previously demonstrated. Naturally, Curraleiro Pé-duro animals had greater levels of CD4, CD8 and γδ lymphocytes (p<0.05). In response to vaccine, Curraleiro Pé-duro showed greater ability to respond specifically to BCG, generating resistance profile (Th1), evidenced by greater number of antigen specific CD4+ cells producing IFN-γ (p<0.05) and also higher tuberculin skin test reaction (p<0.05). Additionally, vaccinated Curraleiro Pé-duro calves had higher CD4 cells numbers than both Nellore control (p<0.05) and vaccinated groups (p<0.05). Curraleiro Pé-duro calves' higher basal lymphocytes blood level and stronger response in both IFN-γ and tuberculin skin test parameters probably play a positive role on protection/resistance to Mycobacterium bovis.

Distribution of immune response cells in the pelvic urethra and the prepuce of rams

The pathogens of the reproductive system in the male can penetrate and establish by ascending route, from to the prepuce to the urethra, accessory glands, epididymis and testicles. The aim of this paper is determine the distribution and number of cells involved in the immune response in prepuce and pelvic urethra of rams, without apparent clinical alterations in testicle, epididymis and prepuce. The distribution of some of the cells involved in the immune response at the level of the prepuce and the pelvic urethra was quantified in four one-year-old rams seronegative for B. ovis and A. seminis and without apparent lesions in the testicles, the epididymis, and the prepuce. At the moment of slaughter, samples were taken from the preputial fornix and the pelvic urethra and placed in 10% formalin and under freezing conditions. CD4, CD8, WC1, CD45RO, CD14 and CD1b cells were demonstrated by immunohistochemistry, and immunoglobulin-containing cells (ICC) of the IgA, IgG and IgM classes were demonstrated by immunofluorescence. The labeled cells present in the mucosa of both organs were counted with an image analyzer. The total number of cells was compared between both tissues and differentially between the epithelium and the connective tissue of the mucosa. Significant differences were found in the total number of CD4, CD45RO, and WC1 lymphocytes, in CD14 macrophages, and CD1b dendritic cells, with mean values being greater in the fornix than in the urethra (p<0.05) in all cases. Only dendritic cells were found in the prepuce. No differences were found in the number of CD8 lymphocytes between both organs. The ratio between each cell type in the connective and the intraepithelial tissues and between organs was 10/1 for CD4 in the fornix (p<0.05), against 7/1 in the urethra (p<0.05), while CD8 had a 1/1 distribution in both mucosae. The WC1 ratio was 5/1 in both mucosae (p<0.05). CD45RO labeling was 19/1 in the prepuce (p<0.05) and 1/1 in the urethra. IgA-containing cells did not show differences in the total number of cells in both tissues. In the urethra, no IgG-containing cells were observed and IgM-containing cells were scarce; in contrast, both cell types were present in the prepuce, in amounts greater than in the urethra (p<0.05). IgA-, IgG-, and IgM-containing cells were located in both organs in the mucosal connective tissue. The presence of antigen-presenting cells, macrophages, and dendritic cells, as well as of lymphocytes CD4, CD8 TCR γδ (WC1), IgA-, IgG and IgM positive cells, and CD45RO cells suggests that both mucosae may behave as inductive and effector sites for the mucosal immune response.

Avaliação imunofenotípica de subpopulações linfocitárias no sangue do cordão umbilical e periférico de suínos neonatos (Sus scrofa)

Considerando a importância do uso do sangue do cordão umbilical como fonte potencial de células tronco hematopoiéticas e o uso do suíno doméstico (Sus scrofa) como modelo para pesquisas biomédicas em medicina regenerativa, e por outro lado, visando dar um contributo sobre a quantificação das subpopulações linfocitárias no sangue do cordão umbilical e periférico, objetivou-se quantificar as células CD4+, CD5+ e CD8+ nas amostras de sangue de suínos neonatos. Analisaram-se as amostras do sangue do cordão umbilical e periférico de 48 leitões de linhagem Topigs, provenientes de porcas hígidas, inseminadas artificialmente e de parto natural. Foram coletadas amostras de sangue do cordão umbilical e periférico no momento do nascimento, por meio de venopunção da veia umbilical e seio venoso retro-oftálmico, respectivamente. As quantificações imunofenotípicas de células CD4+, CD5+ e CD8+ foram obtidas por citometria de fluxo. Os valores médios obtidos para as contagens das células CD4+, CD5+ e CD8+ do sangue do cordão umbilical e periférico apresentaram-se inferiores aos reportados para o sangue periférico de suínos adultos, sugerindo um componente imunológico imaturo. A proporção CD4+:CD8+ obtida no sangue do cordão umbilical (3,2±1,2%) e no sangue periférico (3,2±1,7%) ilustrou a predominância dos linfócitos TCD4+ com relação aos TCD8+. A quantidade relativa de células CD4+ e CD8+ no sangue do cordão umbilical e periférico foi de 1,37±0,86% e 1,15±0,57%, respectivamente.

Regulation of lymphocyte response in vitro and in vivo

CD4+ T helper (Th) cells have an important role in the defence against diverse pathogens. Th cells can differentiate into several functionally distinct subtypes including Th1 and Th2 cells. Th1 cells are important for eradicating intracellular pathogens, whereas Th2 cells pro¬tect our body against extracellular parasites. However if uncontrolled, Th cells can mediate immunopathology such as asthma or allergies, but inappropriate Th response can also lead to autoimmune diseases such as multiple sclerosis or type 1 diabetes. Deeper knowledge of the regulation of the lymphocyte response both in vitro and in vivo is important for un¬derstanding the pathogenesis of immune-mediated diseases and microbe-host interactions. In the work presented in this thesis, the first goal was to elucidate the role of novel factors, PIM kinases and c-FLIP in the regulation of human Th cell differentiation. The oncogenic serine-threonine kinases of the PIM family were shown to be preferentially expressed in Th1 cells and in addition, by using RNA interference, they were also shown to be positive regulators of Th1 differentiation. The PIM depletion experiments suggest that PIM kinases promote the expression of the hallmark cytokine of Th1 cells, IFNγ, and influence the IL12/STAT4 pathway during the early Th1 cell differentiation. In addition to cytokine and T cell receptor (TCR) induced pathways, caspase activity has been shown to regulate Th cell proliferation. In the work presented in this thesis, the two isoforms of the caspase regulator protein, c-FLIP, were shown to be differentially ex¬pressed in Th1 and Th2 cells. Both of the isoforms were up-regulated in response to TCR activation, but the expression of the short isoform was selectively induced by IL4, the Th2 inducing cytokine. Furthermore, the c-FLIP isoforms had distinct and opposite roles during the early differentiation of Th1 and Th2 cells. The knockdown of the long isoform of c-FLIP led to the induction of Th1 marker genes, such as IFNγ and TBET, whereas the depletion of c-FLIP short down-regulated Th2 marker genes IL-4 and GATA3. The third goal was to elucidate the gene expression profiles characterizing the T- and B-lymphocyte responses in vivo during experimental infection by intracellular bacte¬rium Chlamydia pneumoniae. Previously, it has been shown that CD8+ and CD4+ T cells are important for the protection against Chlamydia pneumoniae. In this study, the analysis revealed up-regulation of interferon induced genes during recurrent infection underlining the importance of IFNγ secreted by Th1 and CD8+ T cells in the protection against this pathogen. Taken together, in this study novel regulators of Th cell differ¬entiation were discovered and in addition the gene expression profiles of lymphocytes induced by Chlamydia pneumoniae infection were characterized.

Inflammatory and anti-inflammatory effects of soybean agglutinin

Soybean agglutinin (SBA) lectin, a protein present in raw soybean meals, can bind to and be extensively endocytosed by intestinal epithelial cells, being nutritionally toxic for most animals. In the present study we show that SBA (5-200 µg/cavity) injected into different cavities of rats induced a typical inflammatory response characterized by dose-dependent exudation and neutrophil migration 4 h after injection. This effect was blocked by pretreatment with glucocorticoid (0.5 mg/kg) or by co-injection of N-acetyl-galactosamine (100 x [M] lectin), but not of other sugars (100 x [M] lectin), suggesting an inflammatory response related to the lectin activity. Neutrophil accumulation was not dependent on a direct effect of SBA on the macrophage population since the effect was not altered when the number of peritoneal cells was increased or decreased in vivo. On the other hand, SBA showed chemotactic activity for human neutrophils in vitro. A slight increase in mononuclear cells was observed 48 h after ip injection of SBA. Phenotypic analysis of these cells showed an increase in the CD4+/CD8- lymphocyte population that returned to control levels after 15 days, suggesting the development of an immune response. SBA-stimulated macrophages presented an increase in the expression of CD11/CD18 surface molecules and showed some characteristics of activated cells. After intravenous administration, SBA increased the number of circulating neutrophils and inhibited in a dose-dependent manner the neutrophil migration induced by ip injection of carrageenan into peritoneal cavities. The co-injection of N-acetyl-galactosamine or mannose, but not glucose or fucose, inhibited these effects. The data indicate that soybean lectin is able to induce a local inflammatory reaction but has an anti-inflammatory effect when present in circulating blood

The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection

Cellular immune responses are a critical part of the host's defense against intracellular bacterial infections. Immunity to Brucella abortus crucially depends on antigen-specific T cell-mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. T lymphocytes that proliferate in response to B. abortus were characterized for phenotype and cytokine activity. Human, murine, and bovine T lymphocytes exhibited a type 1 cytokine profile, suggesting an analogous immune response in these different hosts. In vivo protection afforded by a particular cell type is dependent on the antigen presented and the mechanism of antigen presentation. Studies using MHC class I and class II knockout mice infected with B. abortus have demonstrated that protective immunity to brucellosis is especially dependent on CD8+ T cells. To target MHC class I presentation we transfected ex vivo a murine macrophage cell line with B. abortus genes and adoptively transferred them to BALB/c mice. These transgenic macrophage clones induced partial protection in mice against experimental brucellosis. Knowing the cells required for protection, vaccines can be designed to activate the protective T cell subset. Lastly, as a new strategy for priming a specific class I-restricted T cell response in vivo, we used genetic immunization by particle bombardment-mediated gene transfer

Immunologic patterns associated with cure in human American cutaneous leishmaniasis

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-g) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-g and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of IFN-g and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response