Single-nucleotide polymorphisms in 3'-untranslated region inducible costimulator gene and the important roles of miRNA in alopecia areata.

Background: Alopecia areata (AA) spares the stem cell compartment and attacks only the base of the hair follicle, which is surrounded by infiltrating lymphocytes. AA is associated with polymorphisms in immune-related genes and with decreased function of CD4+CD25+ T regulatory (Treg) cells. Treg function is modulated by the costimulatory molecules, like inducible costimulator (ICOS) that are crucial in orienting T cell differentiation and function so that they strongly impact on the immunologic decision between tolerance or autoimmunity development. Objective: The aim of our study was to investigate the possible association of AA with single-nucleotide polymorphisms (SNP) present in the ICOS 3'-untranslated region (3'UTR) region and to elucidate how SNPs modulate ICOS gene expression by affecting miRNA binding sites. Methods: This is a case-control study performed in 184 patients with AA and 200 controls. ICOS gene and miRNA expression were analyzed by real-time polymerase chain reaction. Results: The genotype carrying the rs4404254(C) [p = 0.012, OR (95% CI): 0.5 (0.3-0.8)] and rs4675379(C) [p = 0.015, OR (95% CI): 0.3 (0.1-0.8)] 3' UTR alleles was more frequently observed in AA patients than in controls and correlated with a reduced ICOS expression. miR-1276 significantly suppressed ICOS expression by binding to the 3'UTR of ICOS mRNA. Also, we observed that, miR-101 and miR-27b are upregulated, while miR-103 and miR-2355-3p are downregulated in peripheral blood mononuclear cells of AA patients compared to controls. Conclusion: Our data show that rs4404254 and rs4675379 SNPs of ICOS gene are associated with AA and also reveal that the presence of rs4404254 polymorphism correlates with ICOS post-transcriptional repression by microRNA binding.

Increased frequency of interleukin-4 and reduced frequency of interferon-γ and IL-17-producing CD4+ and CD8+ cells in scleromyxedema.

BACKGROUND: Little is known about the pathogenesis of scleromyxedema, a life-threatening fibromucinosis disease with immunological dysregulation. OBJECTIVES: To investigate on T-cell phenotype, function and cytokine biology in search of new insights supporting the immunopathogenesis of the disease. METHODS: We analysed the frequency of circulating lymphocyte subsets, the T-cell maturation stage, the generation of antigen-specific T-cell lines and T-cell cytokine secretion. RESULTS: The analysis of T-cell maturation stage and the TCR spectratyping findings revealed that scleromyxedema patients showed clear immunological signs of long-lasting immune system activation and stimulation leading to a skewed T-cell repertoire. Moreover, these analyses showed that both CD4+ and CD8+ T cells from scleromyxedema patients have a profound deficiency (even after stimulation) relatively to the production of IFN-γ and IL17 with respect to healthy donor control cells, while they are massively skewed towards IL4 secretion after stimulation. CONCLUSIONS: Our data indicate that a chronic Th2-skewed T-cell response against an unknown target antigen leading to abnormally high IL4 secretion, a pro-fibrotic cytokine, is a main immunological hallmark of scleromyxedema patients. These results, never reported before, may have a translational therapeutic value due to the availability of anti-IL4 agents such as dupilumab.

Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib.

The activation of Nrf2 has been demonstrated to play a crucial role in cancer cell resistance to different anticancer therapies. The inhibition of proteasome activity has been proposed as a chemosensitizing therapy but the activation of Nrf2 could reduce its efficacy. Using the highly chemoresistant neuroblastoma cells HTLA-230, here we show that the strong reduction in proteasome activity, obtained by using low concentration of bortezomib (BTZ, 2.5 nM), fails in reducing cell viability. BTZ treatment favours the binding of Nrf2 to the ARE sequences in the promoter regions of target genes such as heme oxygenase 1 (HO-1), the modulatory subunit of γ-glutamylcysteine ligase (GCLM) and the transporter for cysteine (x-CT), enabling their transcription. GSH level is also increased after BTZ treatment. The up-regulation of Nrf2 target genes is responsible for cell resistance since HO-1 silencing and GSH depletion synergistically decrease BTZ-treated cell viability. Moreover, cell exposure to all-trans-Retinoic acid (ATRA, 3 µM) reduces the binding of Nrf2 to the ARE sequences, decreases HO-1 induction and lowers GSH level increasing the efficacy of bortezomib. These data suggest the role of Nrf2, HO-1 and GSH as molecular targets to improve the efficacy of low doses of bortezomib in the treatment of malignant neuroblastoma.

Single nucleotide polymorphisms in the promoter regions of Foxp3 and ICOSLG genes are associated with Alopecia areata.

Alopecia areata (AA), an autoimmune disease affecting anagen stage hair follicles, is associated with polymorphisms in immune-related genes and with decreased number of CD4+ CD25+ T regulatory cells (Treg). Treg function is modulated by the forkhead box protein 3 (FOXP3) transcription factor and by inducible costimulator (ICOS), through interaction with the relative ligand, ICOSLG, whose genes are polymorphic. The aim of the study was to investigate whether specific single nucleotide polymorphisms (SNPs) of the rs2294020 FOXP3 and/or rs378299 ICOSLG genes may be associated with AA. A case-control study was performed in 120 AA patients and 84 controls. SNPs were analyzed by gene sequencing. FOXP3 and ICOSLG gene expressions were analyzed by real-time PCR. Increased frequencies of the genotype carrying the FOXP3 rs2294020-3675(A) [P = 0.002, OR (95 % CI): 2.55 (1.2-2.7)] or the ICOSLG rs378299-509(C) [P = 0.01, OR (95 % CI): 2.21 (1.1-2.6)] allelic variants were observed in AA patients than in controls. The genotype carrying the combination of the FOXP3 rs2294020-3675(A) and ICOSLG rs378299-509(C) allelic variants with the HLA DQB1*03 allele was more frequently present in AA patients than in controls (P = 0.04). The presence of the FOXP3 rs2294020-3675(A) or the ICOSLG rs378299-509(C) allelic variant was associated with reduced relative gene expression in AA patients. These data suggest that rs2294020 SNP of FOXP3 gene and rs378299 SNP of ICOSLG gene are associated with AA and with a reduced expression of the FOXP3 and ICOSLG genes in alopecia patients.

MF-59 adjuvant influence on the functions of gammadelta T cells in HIV-1+ adults immunized with influenza seasonal vaccine.

INTRODUCTION: We previously reported that in HIV-1 infected patients circulating Vdelta1 T lymphocytes (Vdelta1) increase and proliferate in vitro in response to Candida albicans (Ca). Herein, we analysed the effects of MF59 adjuvant on the Vdelta1 T cell responses to hemagglutinin (HA) and Ca in HIV-1 seropositive and seronegative adults after influenzal vaccine, to clarify th molecular mechanisms triggered in vivo by an adjuvanted vaccine against influenza virus. MATERIALS AND METHODS: 58 seropositive (HIV-1+) and 48 seronegative (HIV-1-) subjects received influenzal vaccines containing or not the MF59 adjuvant. The follow-up of in vitro T cell proliferation and cytokine production (IL-17A, IL-22, IL-23, IL-6) to HA and Ca antigens were performed at different time points (at basal time and after 30 and 90 days from vaccination) by cytofluorimetric approaches. RESULTS: We confirmed that in HIV-1 infected individuals the Vdelta1 T cell subset is expanded in HIV-1 infected individuals and moreover the number of circulating Vdelta1 Tcells significantly enhanced in all HIV-1+ subjects on day 90 after influenza vaccination. Regard the follow-up of proliferative responses, the increments of CD3+ response to HA and Vdelta1 T cells to Ca in HIV-1+ individuals were detectable earlier on day 30 for MF59-vaccinated patients, instead on day 90 post-vaccination in HIV(+)-vaccinated without MF59 adjuvant. Of note, production of lL-17A and IL-22, two cytokines with anti-fungal activity, in response to Ca was enhanced (for IL-17A) or restored (for IL-22) by vaccination in HIV-1+ donors, mainly using the MF59-adjuvanted vaccine. Moreover, after vaccination IL-23 and IL-6 production increased in response to HA in the HIV+ and HIV- groups vaccinated with MF59 adjuvant. CONCLUSIONS: We suggest that in HIV-1 infected patients the circulating Vdelta1 T lymphocytes reactive to Ca upon challenge with influenza virus vaccine receive an activating/enhancing signal mediated by cytokines triggered by the boost with HA antigen particularly in presence of MF59 adjuvant.

Peripheral TH-17 cells in children with allergic rhinitis: preliminary report.

Th17 is a subset of T helper lymphocytes and exerts pro-inflammatory activities. Recently, it has been reported that serum IL-17 levels are high in the most severe patients with birch allergy studied both outside and during the pollen season. This study aims to compare the frequency of peripheral IL-17-producing T cells in children with allergic rhinitis and in healthy controls. Ten children with allergic rhinitis and 5 healthy non-allergic subjects were evaluated. Th17 were evaluated by intracellular staining in ex-vivo T cell compartment. Ex- vivo PBMNC evaluation showed that allergic patients had higher frequencies of IL-17 producing T cells, both concerning CD4+ and CD8+ cells. In particular, there is a subset co-expressing IL-17 and IFN-gamma both for CD4+ and CD8+ cells. In conclusion, this preliminary study suggests a possible role of Th-17 cells in the response to allergens in children.

The role of AIRE polymorphisms in melanoma.

Polymorphisms of AIRE, a transcription factor that up-regulates intrathymic expression of tissue-specific antigens including melanoma-associated antigens (MAAs), may variably affect the selection of MAAs-specific thymocytes, generating T-cell repertoires protecting or predisposing individuals to melanoma. We found that AIRE single nucleotide polymorphisms (SNPs) rs1055311, rs1800520 and rs1800522 were significantly more frequent in healthy subjects than in melanoma patients, independently from sex, age and stages of melanoma. The presence of these SNPs was associated with increased frequency of two T-cell clonotypes specific for MAGE-1 linking their protective effect to selection/expansion of MAA-specific T cells. Interestingly, mRNA transcribed on the rs1800520 SNP showed increased free energy than the wild type suggesting that its reduced stability may be responsible for the different activity of the polymorphic AIRE molecule. This finding may contribute at identifying subjects with increased risk of developing melanoma or patients with melanoma that may take benefit from immunotherapy.

Relevance of CD38 expression on CD8 T cells to evaluate antiretroviral therapy response in HIV-1-infected youths.

Surrogate markers for monitoring immuno-virological discordant responders, in addition to plasma viral load and CD4 cells, are still lacking. We assessed the diagnostic utility of CD38 expression on CD8 T cell assay, alone or in association with lymphocyte proliferation to mycotic antigens, in evaluating antiretroviral response. 28 vertically HIV-infected youths, 21 HAART- and seven 2 nucleotide reverse transcriptase inhibitors-treated, were enrolled in a retrospective study. Responders (57.1%) and non-responders (42.9%) to stable antiretroviral therapy for a minimum of 6 months, on the basis of viral load and CD4 T cells, comprehensively evaluated by CD38 expression on CD8 T lymphocytes [measured as CD38 antibody bound per CD8 T cell (CD38 ABC) and %CD38+ of total CD8 T cells (%CD38/CD8)] and lymphocyte proliferation to P. jiroveci, C. albicans, C. neoformans, A. fumigatus at a single time point after treatment, were selected. CD38 expression > or =2401 CD38 ABC and > or =85% CD38/CD8 cut-off points, accurately discriminates responders versus non-responders, both measures resulting in 75.0% (CI 42.8-94.5) sensitivity (identification of non-responder) and 93.8% (CI 69.8-99.8) specificity (identification of responder), when considered as single assays. The association '> or =2401 CD38 ABC or > or =85% CD38/CD8' improved sensitivity to 83.3% (CI 51.6-97.9), while the association '<2401 CD38ABC (or <85% CD38/CD8) and lymphoproliferative response positive to > or =2 tested organisms' improved specificity to 100% (CI 79.4-100). In conclusions, CD38 expression and mycotic antigen-specific T-cell proliferation may be used as additional parameters to existing criteria to evaluate antiretroviral response in immuno-virological discordant patients.

Conserved T cell and natural killer cell function in treatment-experienced adults receiving tenofovir plus didanosine as nucleoside reverse transcription inhibitor backbone.

Anti-retroviral treatment (ART) usually results in efficient control of virus replication and in immune reconstitution. Among potential adverse effects, impairment of immune responses in terms of CD4(+) T cell counts has been attributed to some ART regimens, as with didanosine-tenofovir. We studied the functional integrity of adaptive and innate immunity during didanosine-tenofovir-containing ART. Two groups of extensively pretreated patients completing at least 48 weeks of ART containing either lamivudine-didanosine (n = 21) or tenofovir-didanosine (n = 25) were identified. In addition to standard clinical immune and virological parameters, we performed a flow cytometric analysis of natural killer (NK) cells, of memory and naive CD4(+) T cells and of T cell receptor alphabeta(+) T cells co-expressing inhibitory NK receptors. Functional analysis consisted in specific and total interferon-gamma production by NK cells and of recall antigen proliferation of peripheral blood mononuclear cells. Comparable clinical immunological reconstitution and virological control were confirmed in the two groups of patients in the absence of clinically relevant adverse effects. The proportion of CD4(+)CD45RA(+) T cells and of functionally inhibited killer immunoglobulin-like receptor T cell receptor alphabeta(+) cells, the proliferation to recall antigens as well as NK cell phenotype and function as determined by interferon-gamma production in patients treated with tenofovir-didanosine were comparable to those treated with a different regimen. Thus, no differences in functional innate or adaptive immune reconstitution are detected in drug-experienced human immunodeficiency virus-infected patients on tenofovir-didanosine nucleoside reverse transcription inhibitor regimens.

Ebastine increases IFN-gamma production in patients with persistent allergic rhinitis.

Nasal obstruction is a leading symptom in patients with allergic rhinitis and depends on inflammation characterized by Th2 polarization. Thus, IFN-gamma is typically deficient in allergic patients. It has been previously reported that ebastine is able to reduce Th2-dependent cytokines. The aim of this study is to preliminarily evaluate IFN-gamma production by peripheral blood mononuclear cells (PBMNC) and clinical changes after a treatment with lyophilized ebastine in patients with persistent allergic rhinitis (PER). Ten patients with PER were evaluated, 7 males and 3 females (mean age 32.4 +/- 6.2 years), all of whom received lyophilized ebastine (20 mg/daily) for 3 weeks. Total nasal symptom score (TSS), subjective evaluation score by visual analogue scale (VAS), and rhinomanometry were evaluated in all subjects before and after treatment. IFN-gamma production by peripheral blood mononuclear cells (PBMNC) was evaluated using different stimuli, in un-treated and ebastine-treated allergic patients by ELISPOT. Ebastine treatment induced significant increase of IFN-gamma production stimulated by grasses (p<0.0001) and Dermatophagoides farinae (p=0.0015). This effect was significantly related with TSS and VAS improvement after treatment (p=0.0038 and 0.004 respectively). In conclusion, this preliminary study demonstrates the effectiveness of ebastine treatment in increasing IFN-gamma production. The clinical relevance of this study is that the clinical improvement is related to the immunologic activity.

Serum interleukin-17 levels are related to clinical severity in allergic rhinitis.

BACKGROUND: T helper (Th)-17 cells are a subset of T helper lymphocytes that exert regulatory activities. Recently, it has been reported that serum interleukin (IL)-17 levels are high in the most severe cases of birch allergy studied outside the pollen season. OBJECTIVE: The aim of this study was to investigate a possible relationship between serum IL-17 levels and clinical parameters in patients with allergic rhinitis studied during the pollen season. METHODS: In all, 56 patients with persistent pollen-induced allergic rhinitis were evaluated during the pollen season. Serum IL-17 levels were evaluated by enzyme-linked immunosorbent assay. Symptoms were assessed by visual analogue scale, drug use was monitored and peripheral eosinophils were counted. RESULTS: Serum IL-17 levels were significantly related to clinical symptoms, drug use and peripheral eosinophil counts (P = 0.0001 for all). CONCLUSION: This study provides evidence that serum IL-17 level assessment might be considered to classify allergy severity.

Sublingual immunotherapy provides an early increase of interferon-gamma production.

Allergic rhinitis (AR) is characterized by Th2 polarized immune response. Allergen-specific subcutaneous immunotherapy may restore a physiologic Th1 profile. However, there are few studies investigating the immunological effects of sublingual immunotherapy (SLIT). The aim of this study is to investigate whether a pre-seasonal SLIT course could affect IFN-gamma production. Forty-four AR patients with pollen allergy assumed pre-seasonal SLIT for 3 months. IFN-gamma-specific producing cells were assessed by cytokine ELISPOT before and 3 months after the beginning of SLIT. Visual analogue scale (VAS) for symptoms and medication score was also evaluated. The frequency of IFN-gamma-specific producing cells significantly increased after SLIT (p<0.01), and this increase was significantly associated with improvement of both symptoms (p<0.001) and medication use (p<0.01). In conclusion, these results may be considered clinically relevant as SLIT treatment may induce a quick IFN- gamma response that is related to clinical improvement.

Assessment of humoral and cell-mediated immunity against Bordetella pertussis in adolescent, adult, and senior subjects in Italy.

Humoral and cell-mediated immunity (CMI) against B. pertussis was assessed in a sample of adolescent, adult and senior subjects distributed in five different geographical areas in Italy. Most (99.1%) subjects had IgG anti-pertussis toxin (PT) antibodies exceeding the minimum detection level [> or = 2 ELISA units (EU)/ml]. There were no significant differences between the genders; 6.2% samples recorded titres > or = 100 EU/ml. CMI was positive [stimulation index (SI) > or = 5] against PT in 39.0% of all samples. This study suggests that B. pertussis continues to circulate in age groups that have been previously considered to be uninvolved in the circulation of this pathogen and that adolescent and adult pertussis boosters may be of value in these populations. Nevertheless, over the last 10 years, large increases in vaccination coverage rates have contributed to reduce the spread of the aetiological agent, especially in the immunized population.

Safety and immunogenicity of two influenza virus subunit vaccines, with or without MF59 adjuvant, administered to human immunodeficiency virus type 1-seropositive and -seronegative adults.

The objective of this study was to evaluate and compare both the safety and tolerability and the humoral and cell-mediated immune responses for two influenza virus subunit vaccines, one with MF59 adjuvant (Fluad) and one without an adjuvant (Agrippal), in healthy and in human immunodeficiency virus type 1 (HIV-1)-infected adult individuals. To achieve this aim, an open, randomized, comparative clinical trial was performed during the 2005-2006 season. A total of 256 subjects were enrolled to receive one dose of vaccine intramuscularly. Blood samples were taken at the time of vaccination and at 1 and 3 months postvaccination. A good humoral antibody response was detected for both vaccines, meeting all the criteria of the Committee for Medical Products for Human Use. After Beyer's correction for prevaccination status, Fluad exhibited better immunogenicity than Agrippal, as shown from the analysis of the geometric mean titers, with significant differences for some virus strains; however, no definitive conclusions on the clinical significance of such results can be drawn, because the method used to estimate antibody response is currently nonstandard for influenza virus vaccines. Significant induction of an antigen-specific CD4+ T-lymphocyte proliferative response was detected at all time points after immunization, for both the vaccines, among HIV-1-seronegative subjects. This was different from what was observed for HIV-1-infected individuals. In this group, significance was not reached at 30 days postvaccination (T30) for those immunized with Agrippal. Also when data were compared between treatment groups, a clear difference in the response at T30 was observed in favor of Fluad (P = 0.0002). The safety profiles of both vaccines were excellent. For HIV-1-infected individuals, no significant changes either in viremia or in the CD4+ cell count were observed at any time point. The results showed good safety and immunogenicity for both vaccines under study for both uninfected and HIV-1-infected adults, confirming current recommendations for immunization of this high-risk category.

Non-antigen-specific CD8(+) T suppressor lymphocytes in diseases characterized by chronic immune responses and inflammation.

Recent studies on regulatory lymphocytes demonstrate that CD8(+) T suppressor (Ts) cells may have great relevance in controlling immune system homeostasis and avoiding development of chronic inflammatory diseases. Among the three subpopulations of CD8(+) Ts cells so far recognized in humans, the type 2 (non-antigen-specific) cell is characterized by the capacity to inhibit both T cell proliferation and cytotoxic T lymphocyte activity through secretion of soluble factors. Previous work has shown the impairment of in vitro generation of type 2 CD8(+) Ts cells from the peripheral blood of relapsed patients with multiple sclerosis, systemic lupus erythematosus, or systemic sclerosis. Here, similar findings are demonstrated for patients with human immunodeficiency virus or chronic hepatitis C virus infection. Furthermore, the presence of type 2 CD8(+) Ts cells infiltrating diseased tissues in patients with autoimmune thyroiditis or cancer is shown. Collectively, these findings suggest that type 2 CD8(+) Ts cells may be involved in the control of pathologic chronic immune responses, contributing in some cases to the pathogenesis of the disease.

Sublingual specific immunotherapy reduces PBMC proliferations.

BACKGROUND: Subcutaneous specific immunotherapy has been demonstrated capable of inducing T regulatory response. There is few evidence concerning immunological changes induced by sublingual immunotherapy. OBJECTIVE: The aim of this study was to evaluate T cell proliferation in subjects successfully treated with SLIT for HDM. METHODS: PBMCs were isolated from patients after at least 3 years of successful HDM SLIT and from matched untreated allergic and healthy control subjects. After 3 and 6 days of in vitro stimulation with PHA, Candida albicans, Dermatophagoides farinae, grasses, Parietaria judaica, and cat, proliferation. RESULTS: Subjects treated with SLIT showed significant reduction of proliferation induced by Candida albicans, Parietaria, and grasses in comparison with untreated atopics (p=0.0002, 0.0033, and 0.009 respectively). CONCLUSION: This pilot study confirms reduced T cell proliferation in allergic subjects treated with SLIT.

Non-antigen specific CD8+ T suppressor lymphocytes.

The homeostasis of peripheral immune system function is maintained by the activity of regulatory lymphocytes. Among these cells, a subset of CD8+CD28- T suppressor lymphocytes has recently been characterized for the capacity to mediate their effects without antigen restriction. These non-antigen-specific CD8+ T suppressor lymphocytes originate from circulating CD8+CD28- T lymphocytes after stimulation with interleukin-2 and interleukin-10. CD8+ suppressor cells inhibit both antigen-specific CD4+ T cell proliferation and cellular cytoxicity through secretion of cytokines such as interferon-gamma, interleukin-6, and interleukin-10. The function of CD8+ suppressor cells is impaired in patients with systemic lupus erythematosus in relapse as well as in patients with systemic sclerosis with disease progression, suggesting the involvement of CD8+ suppressor cells in the pathogenesis of autoimmune diseases. Interestingly, CD8+ suppressor cells have been found among tumor-infiltrating lymphocytes, which could be related to tumor-induced-immunosuppression. Failure to generate CD8+ suppressor cells from the peripheral blood is frequently observed in HIV-infected patients. It remains to be clarified whether this phenomenon is due to depletion and/or functional impairment of this cell subset or to their compartmentalization in peripheral tissues and immunocompetent organs where they could contribute to the induction of immunodeficiency.

Modifications of immunological and neuro-endocrine parameters induced by antiorthostatic bed-rest in human healthy volunteers.

AIM: Space flight has profound effects on immunological and neuroendocrine parameters. Microgravity plays a major role in the induction of these changes. The aim of the present study was the evaluation on ground of the effects induced by antigravitary posture on immune and neuroendocrine functions. METHODS: Eight healthy male volunteers (mean age 24+/-1 years) were maintained in antigravitary posture (-10 degrees) for 72 hours. Four of them were also maintained in supine posture for 72 hours as controls. The following immunological and neuroendocrine parameters have been analysed: peripheral white blood cells count, CD11b integrin expression and H(2)O(2) production by neutrophils, lymphocyte and monocyte phenotype, intracytoplasmic cytokine (IFN-gamma, TNF-alpha and IL-4) pattern, lymphocyte proliferation to mitogens and antigens, cortisol, ACTH, catecholamines, GH, LH, prolactin and testosterone plasma levels. RESULTS: In subjects maintained in antigravitary posture, norepinephrine, dopamine, cortisol, ACTH, GH and prolactin plasma levels increased whereas H(2)O(2) production by neutrophils, lymphocyte proliferation, NK cells number and intracytoplasmic IFN-g expression decreased. No significant modifications were observed in subjects maintained in supine posture. CONCLUSION: The results of this study indicate that several neuroendocrine and immunological parameters are modulated by a prolonged antigravitary posture on ground and may negatively affect astronauts defenses against pathogens during space flights.

Preservation of clonal heterogeneity of the Pneumocystis carinii-specific CD4 T cell repertoire in HIV infected, asymptomatic individuals.

The loss of CD4 lymphocytes in HIV disease associates with opportunistic infections. Since diverse CD4 T cell clones respond to an opportunistic pathogen, we asked whether CD4 depletion deletes selected clones in the repertoire (vertical depletion) or it affects all clones by reducing the cell number in each progeny without affecting the overall number of clones (horizontal depletion). Understanding this point may help explain the mode of CD4 depletion and the mode of immunoreconstitution after therapy. Therefore we examined the CD4 T cell repertoire specific for Pneumocystis carinii, a relevant opportunistic pathogen in AIDS, in HIV-infected, asymptomatic individuals. We identified two patients of 36 asymptomatics for lack of proliferation to P. carinii, suggesting selective depletion of specific CD4 cells. To investigate clonal heterogeneity of P. carinii-responsive CD4 lymphocytes, specific CD4 T cell lines were generated and studied by TCR BV gene family usage and CDR3 length analysis (spectratyping). Clonal heterogeneity was similar in antigen-specific CD4 lines generated from P. carinii non-responding HIV seropositives and from controls. Thus, despite undetectable response to the pathogen, residual specific cells probably prevent overt infection and, when expanded in vitro, exhibit a clonal diversity similar to normal controls. These findings suggest a horizontal, rather than vertical, depletion in these asymptomatic patients.