Peripheral blood mononuclear cells immunophenotyping in pulmonary tuberculosis patients before and after treatment

Tuberculosis (TB) is a lung disease caused by Mycobacterium tuberculosis. The interaction between the bacillus and the host may lead to a protective cellular immune response. In the present study, we propose the "in vitro" evaluation of this cellular immune response in patients with tuberculosis before and after chemotherapic treatment. Eleven patients with TB and 9 asymptomatic subjects with tuberculin skin test negative (TST-) (purified protein derivative (PPD) 相似文献   

CD4(+) T cell levels are decreased during active CMV infection in kidney transplant recipients

The numbers of T lymphocytes and T cell subsets (CD2(+), CD3(+), CD4(+), CD8(+)), activated T cells (CD26(+)), B cells (CD19(+)), granulocytes (CD15(+)) and natural killer cells (CD16/56) were monitored by flow cytometry in 79 kidney transplant recipients, 35 of whom had cytomegalovirus infection. The percentages of these cells were correlated with viral load, as determined by cytomegalovirus antigenemia. Development of cytomegaloviral infection coincided with a significant reduction in the percentages of CD4(+) (P < 0.005) and CD3(+) (P < 0.05) cells. Monitoring of lymphocyte subsets may provide useful information on immunological events during cytomegaloviral infection.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

NKT cells in the induced sputum of severe asthmatics

To determine whether there was a specific inflammatory process in severe asthmatics, the phenotypic characteristics of induced sputum immune cells were analysed among patients with severe asthma. Twenty-two induced sputa (10 severe asthmatics) were studied. Flow cytometric analysis was performed using immune cells of the sputum and monoclonal antibodies to CD3, CD4, CD8, CD56, CD25, and TCRgammadelta. The number of NKT (CD3(+) CD56(+)) cells was significantly higher in the sputum of severe asthmatics compared with mild asthmatic and healthy control groups (P < .05). CD8(+)CD56(+) cells were the predominant subtype of the increased NKT cells in severe asthmatics. CD3(+)CD56(+)Valpha24(+), TCRgammadelta(+) CD56(+), and CD4(+)CD25(+) T cells were significantly increased in severe asthmatic patients. These results suggest that the immunopathogenesis of severe asthmatics vary between severe and mild asthmatics, and that CD8(+)CD56(+) NKT cells may play an important role in the immunopathogenesis of severe asthma.     

Contribution of exertional hyperthermia to sympathoadrenal-mediated lymphocyte subset redistribution.

The contribution of hyperthermia to the differential leukocytosis of exercise remains obscure. This study examined changes in circulating sympathoadrenal hormone concentrations and patterns of leukocyte and lymphocyte subset (CD3(+), CD4(+), CD8(+), CD19(+), CD3(-)16(+)/56(+)) redistribution during exercise, with and without a significant rise of rectal temperature (T(re)). Ten healthy men [age 26.9 +/- 5.7 (SD) yr, body mass 76.0 +/- 10.9 kg, body fat 13.9 +/- 4.6%, peak O(2) consumption: 48.0 +/- 12.4 ml x kg(-1) x min(-1)] exercised for 40 min (65% peak O(2) consumption) during water immersion at 39 or 18 degrees C. T(re) increased from 37.2 to 39.3 degrees C (P < 0.0001) after 40 min of exercise in 39 degrees C water but was held constant to an increment of 0.5 degrees C during exercise in 18 degrees C water. Application of this thermal clamp reduced exercise-associated increments of plasma epinephrine (Epi) and norepinephrine (NE) by >50% (P < 0.05) and abolished the postexercise increase in cortisol. Thermal clamping also reduced the exercise-induced leukocytosis and lymphocytosis. Multiple regression demonstrated that T(re) had no direct association with lymphocyte subset mobilization but was significantly (P < 0.0001) correlated with hormone levels. Epi was an important determinant of total leukocytes, lymphocytes, and CD3(+), CD4(+), CD8(+), and CD3(-)CD16(+)/56(+) subset redistribution. The relationship between NE and lymphocyte subsets was weaker than that with Epi, with the exception of CD3(-)CD16(+)/56(+) counts, which were positively (P < 0.0001) related to NE. Cortisol was negatively associated with leukocytes, CD14(+) monocytes, and CD19(+) B- and CD4(+) T-cell subsets but was positively related to granulocytes. We conclude that hyperthermia mediates exercise-induced immune cell redistribution to the extent that it causes sympathoadrenal activation, with alterations in circulating Epi, NE, and cortisol.     

The role of tumor cells in the modification of T lymphocytes activity--the expression of the early CD69+, CD71+ and the late CD25+, CD26+, HLA/DR+ activation markers on T CD4+ and CD8+ cells in squamous cell laryngeal carcinoma. Part I

The role of interactions between tumor cells and autologous immunocompetent cells, the impact on the modulation of the activity of T CD4(+) and CD8(+) lymphocytes, as well as the influence on the regulation and determination of antitumor cellular immune response in patients with head and neck squamous cell carcinomas (HNSCC) is not completely clear. The aim of this study was to analyze early and late activation antigens expression on T cells subpopulations modified under the influence of the presence of cancer cells to investigate the regulatory mechanisms of the local cellular immune response in carcinoma of the larynx. Cytofluorymetric analysis of the early (CD69(+), CD71(+)) and late activation markers (CD25(+) (high), CD26(+), HLA/DR(+)) expression on T CD3(+)CD4(+) and CD3(+)CD8(+) cells subpopulations in mixed cellular cultures of freshly isolated tumor cells (MLTMC) and non-cancerous normal epithelial cells (MLNCC) with immunocompetent cells was performed in 55 cases of squamous cell laryngeal carcinoma. The whole peripheral blood concentrations of IL-10 and IFN-γ in 21 h and 72 h of experiments were also measured by ELISA. The relationships between the activation markers expression depending on the type of cells used in co-cultures, as well as the level of secreted cytokines, were investigated. Our work has revealed a statistically significant dependence of cytofluorymetric results on the presence of TMC or NCC in mixed cellular cultures. Increased expression of CD69(+), CD71(+) and CD25(+) (high), CD26(+), HLA/DR(+) antigens on T CD3(+)CD4(+) and CD3(+)CD8(+) cells was higher in MLTMC cultures, in comparison with MLNCC. We demonstrated negative significant relationships of IFN-γ and IL-10 secretion with regard to CD4(+)CD69(+), CD8(+)CD69(+), CD4(+)CD71(+), CD8(+)CD71(+) antigens expression in 21 h of experiments without mitogenic stimulation. Furthermore, this study revealed negative significant relationships of IFN-g secretion with regard to CD4(+)HLA/DR(+) and CD8(+)HLA/DR(+) as well as between IL-10 concentration and CD4(+)HLA/DR(+) in trials without PHA stimulation. Our findings have confirmed a key role for tumor cells in determining the function of T cells involved in the immunological processes and impact of neoplastic cells on modulating the activity of T CD4(+) and CD8(+) lymphocytes in laryngeal carcinoma.     

TUNEL and limited immunophenotypic analyses of apoptosis in paucibacillary and multibacillary leprosy lesions

Some mycobacterial infections, such as tuberculosis, are characterized by apoptosis of infected or by-stander mononuclear immune cells. For localized (paucibacillary, PB) and disseminated (multibacillary, MB) leprosy, characterized by polarized Th1-like vs. Th2-like immune responses, respectively, little is known about lesional apoptosis. We analyzed sections of paraffin-embedded, untreated leprosy lesions from 21 patients by an indirect immunofluorescent terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. Some TUNEL (+) PB sections were then reacted with phycoerythrin-conjugated (red) antibodies against T cells, monocytes, or antigen-presenting (Langerhans) cells. TUNEL (+) bodies were detected in 9 of 16 PB lesions (56%) and in 1 of 5 MB lesions (20%). Some TUNEL (+) bodies in PB disease were CD3+ (T cell), as well as CD4+ (T-helper) or CD8+ (T-cytotoxic). Apoptosis characterizes PB and MB leprosy lesions and may be more frequent in PB disease. In PB disease, some TUNEL (+) bodies may derive from T cells.     

Changes in serum cytokine levels in active tuberculosis with treatment

It has been reported that IFN-gamma, TNF-alpha, and IL-12 stimulate, and that IL-10, TGF-beta, and IL-4 suppress the protective immune response against tuberculosis. We aim to evaluate changes in the serum levels of pro and antiinflammatory cytokines in active pulmonary tuberculosis (APTB) and the possible effects of treatment on these changes. Serum IL-12p40, IL-4, IL-10, TNF-alpha, IFN-gamma, and TGF-beta1 levels were determined in 20 APTB cases (group 1) before and 2, 4, and 6 months after therapy. The same parameters were also determined in 9 inactive pulmonary tuberculosis (IPTB) cases (group 2) and 9 healthy controls (HC, group 3). Before treatment, the mean serum IFN-gamma, TNF-alpha, and IL-10 levels in group 1 were statistically higher than those in group 2 (P=.001, P=.024, P=.016, resp) or group 3 (P=.003, P=.002, P=.011, resp). The levels in group 1 decreased significantly after treatment (P=.001 for IFN-gamma, P=.004 for TNF-alpha, P=.000 for IL-10). The serum levels of IL-12p40 were significantly higher in group 1 than in group 3 (P=.012) and decreased insignificantly after treatment. There was no difference in serum IL-4 and TGF-beta1 levels among the groups (P>.05). Because the serum IL-12p40, IL-10, TNF-alpha, and IFN-gamma levels were high in APTB, we believe that these cytokines have important roles in the immune response to Mycobacterium tuberculosis (M tuberculosis). These parameters could be used in follow-up as indicators of the success of APTB therapy.     

CD8(+), alphabeta-TCR(+), and gammadelta-TCR(+) cells in the recipient hematopoietic environment mediate resistance to engraftment of allogeneic donor bone marrow

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of alphabeta-TCR(+), gammadelta-TCR(+), alphabeta- plus gammadelta-TCR(+), CD8(+), or CD4(+) cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4(+) T cells. Mice lacking both alphabeta- and gammadelta-TCR(+) cells engrafted without conditioning, suggesting that both alphabeta- and gammadelta-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8(-) cell was targeted by the CyP conditioning. The CD8(+) cell effector function is mechanistically different from that for conventional T cells, and independent of CD4(+) T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.     

Characterization of rat CD8+ uveitogenic T cells specific for interphotoreceptor retinal-binding protein 1177-1191

The uveitogenic T cells that mediate experimental autoimmune uveitis are commonly assumed to be exclusively CD4(+). In the present study, we showed that, although a panel of long-term cultured rat uveitogenic T cell lines specific for the interphotoreceptor retinal-binding protein peptide, R16, all expressed CD4, approximately 40% of the R16-specific uveitogenic T cells freshly prepared from Ag-immunized rats were CD8(+)alphabetaTCR(+), as demonstrated by CFSE staining. We showed that the expansion of these CD8(+)alphabetaTCR(+) T cells was Ag-specific and that highly purified CD8(+) R16-specific T cells were able to induce uveitis on transfusion into naive rats. Moreover, CD8(+) uveitogenic T cells more readily switched phenotype from, and to, TCR(-)CD8(-)CD4(-) during in vivo or in vitro activation compared with their CD4(+) counterparts. In a previous study, we showed that highly purified CD8(+) myelin oligodendrocyte glycoprotein-specific T cells induced more severe autoimmune encephalomyelitis than the corresponding CD4(+) T cells. In this study, we show that an interphotoreceptor retinal-binding protein peptide consistently activated a high proportion of CD8(+)alphabetaTCR(+) T cells, which were uveitogenic in Lewis rats.     

Characterization of a Mycobacterium tuberculosis peptide that is recognized by human CD4+ and CD8+ T cells in the context of multiple HLA alleles

The secreted Mycobacterium tuberculosis 10-kDa culture filtrate protein (CFP)10 is a potent T cell Ag that is recognized by a high percentage of persons infected with M. tuberculosis. We determined the molecular basis for this widespread recognition by identifying and characterizing a 15-mer peptide, CFP10(71-85), that elicited IFN-gamma production and CTL activity by both CD4(+) and CD8(+) T cells from persons expressing multiple MHC class II and class I molecules, respectively. CFP10(71-85) contained at least two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6). T1 was recognized by CD4(+) cells in the context of DRB1*04, DR5*0101, and DQB1*03, and by CD8(+) cells of A2(+) donors. T6 elicited responses by CD4(+) cells in the context of DRB1*04 and DQB1*03, and by CD8(+) cells of B35(+) donors. Deleting a single amino acid from the amino or carboxy terminus of either peptide markedly reduced IFN-gamma production, suggesting that they are minimal epitopes for both CD4(+) and CD8(+) cells. As far as we are aware, these are the shortest microbial peptides that have been found to elicit responses by both T cell subpopulations. The capacity of CFP10(71-85) to stimulate IFN-gamma production and CTL activity by CD4(+) and CD8(+) cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antituberculosis vaccine.     

Conformational and biochemical differences in the TCR.CD3 complex of CD8(+) versus CD4(+) mature lymphocytes revealed in the absence of CD3gamma

Mature CD4(+) and CD8(+) T lymphocytes are believed to build and express essentially identical surface alphabeta T-cell receptor-CD3 (TCR.CD3) complexes. However, TCR.CD3 expression has been shown to be more impaired in CD8(+) cells than in CD4(+) cells when CD3gamma is absent in humans or mice. We have addressed this paradox by performing a detailed phenotypical and biochemical analysis of the TCR.CD3 complex in human CD3gamma-deficient CD8(+) and CD4(+) T cells. The results indicated that the membrane TCR.CD3 complex of CD8(+) T lymphocytes was conformationally different from that of CD4(+) lymphocytes in the absence of CD3gamma. In addition, CD8(+), but not CD4(+), CD3gamma-deficient T lymphocytes were shown to contain abnormally glycosylated TCRbeta proteins, together with a smaller, abnormal TCR chain (probably incompletely processed TCRalpha). These results suggest the existence of hitherto unrecognized biochemical differences between mature CD4(+) and CD8(+) T lymphocytes in the intracellular control of alphabetaTCR. CD3 assembly, maturation, or transport that are revealed when CD3gamma is absent. Such lineage-specific differences may be important in receptor-coreceptor interactions during antigen recognition.     

NKT cells from normal and tumor-bearing human livers are phenotypically and functionally distinct from murine NKT cells

A major group of murine NK T (NKT) cells express an invariant Valpha14Jalpha18 TCR alpha-chain specific for glycolipid Ags presented by CD1d. Murine Valpha14Jalpha18(+) account for 30-50% of hepatic T cells and have potent antitumor activities. We have enumerated and characterized their human counterparts, Valpha24Vbeta11(+) NKT cells, freshly isolated from histologically normal and tumor-bearing livers. In contrast to mice, human NKT cells are found in small numbers in healthy liver (0.5% of CD3(+) cells) and blood (0.02%). In contrast to those in blood, most hepatic Valpha24(+) NKT cells express the Vbeta11 chain. They include CD4(+), CD8(+), and CD4(-)CD8(-) cells, and many express the NK cell markers CD56, CD161, and/or CD69. Importantly, human hepatic Valpha24(+) T cells are potent producers of IFN-gamma and TNF-alpha, but not IL-2 or IL-4, when stimulated pharmacologically or with the NKT cell ligand, alpha-galactosylceramide. Valpha24(+)Vbeta11(+) cell numbers are reduced in tumor-bearing compared with healthy liver (0.1 vs 0.5%; p < 0.04). However, hepatic cells from cancer patients and healthy donors release similar amounts of IFN-gamma in response to alpha-galactosylceramide. These data indicate that hepatic NKT cell repertoires are phenotypically and functionally distinct in humans and mice. Depletions of hepatic NKT cell subpopulations may underlie the susceptibility to metastatic liver disease.     

Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A

The role of CD8(+) CTL in protection against tuberculosis in human disease is unclear. In this study, we stimulated the peripheral blood mononuclear cells of bacillus Calmette-Guérin (BCG)-vaccinated individuals with live Mycobacterium bovis BCG bacilli to establish short-term cell lines and then purified the CD8(+) T cells. A highly sensitive enzyme-linked immunospot (ELISPOT) assay for single cell IFN-gamma release was used to screen CD8(+) T cells with overlapping peptides spanning the mycobacterial major secreted protein, Ag85A. Three peptides consistently induced a high frequency of IFN-gamma responsive CD8(+) T cells, and two HLA-A*0201 binding motifs, P(48-56) and P(242-250), were revealed within the core sequences. CD8(+) T cells responding to the 9-mer epitopes were visualized within fresh blood by ELISPOT using free peptide or by binding of HLA-A*0201 tetrameric complexes. The class I-restricted CD8(+) T cells were potent CTL effector cells that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombinant vaccinia virus expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold higher frequency of peptide-specific T cells than IFN-gamma ELISPOT assays, indicating functional heterogeneity within the CD8(+) T cell population. These results demonstrate a previously unrecognized, MHC class I-restricted, CD8(+) CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.     

Induction of human papillomavirus-specific CD4(+) and CD8(+) lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16- and 18-positive cervical cancer.

Human papillomavirus (HPV) type 16 (HPV 16) and HPV type 18 (HPV 18) are implicated in the induction and progression of the majority of cervical cancers. Since the E6 and E7 oncoproteins of these viruses are expressed in these lesions, such proteins might be potential tumor-specific targets for immunotherapy. In this report, we demonstrate that recombinant, full-length E7-pulsed autologous dendritic cells (DC) can elicit a specific CD8(+) cytotoxic T-lymphocyte (CTL) response against autologous tumor target cells in three patients with HPV 16- or HPV 18-positive cervical cancer. E7-specific CTL populations expressed strong cytolytic activity against autologous tumor cells, did not lyse autologous concanavalin A-treated lymphoblasts or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL), and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. Cytotoxicity against autologous tumor cells could be significantly blocked by anti-HLA class I (W6/32) and anti-CD11a/LFA-1 antibodies. Phenotypically, all CTL populations were CD3(+)/CD8(+), with variable levels of CD56 expression. CTL induced by E7-pulsed DC were also highly cytotoxic against an allogeneic HLA-A2(+) HPV 16-positive matched cell line (CaSki). In addition, we show that specific lymphoproliferative responses by autologous CD4(+) T cells can also be induced by E7-pulsed autologus DC. E7-specific CD4(+) T cells proliferated in response to E7-pulsed LCL but not unpulsed LCL, and this response could be blocked by anti-HLA class II antibody. Finally, with two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, a marked Th1-like bias (as determined by the frequency of gamma interferon- and interleukin 4-expressing cells) was observable for both CD8(+) and CD4(+) E7-specific lymphocyte populations. Taken together, these data demonstrate that full-length E7-pulsed DC can induce both E7-specific CD4(+) T-cell proliferative responses and strong CD8(+) CTL responses capable of lysing autologous naturally HPV-infected cancer cells in patients with cervical cancer. These results may have important implications for the treatment of cervical cancer patients with active or adoptive immunotherapy.     

CD16+ monocyte-derived macrophages activate resting T cells for HIV infection by producing CCR3 and CCR4 ligands

The CD16(+) monocyte (Mo) subset produces proinflammatory cytokines and is expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unclear. In this study, we investigate the capacity of human CD16(+) and CD16(-) Mo subsets to render resting CD4(+) T cells permissive for HIV replication. We demonstrate that CD16(+) Mo preferentially differentiate into macrophages (Mphi) that activate resting T cells for productive HIV infection by producing the CCR3 and CCR4 ligands CCL24, CCL2, CCL22, and CCL17. CD16(+), but not CD16(-), Mo-derived Mphi from HIV-infected and -uninfected individuals constitutively produce CCL24 and CCL2. Furthermore, these chemokines stimulate HIV replication in CD16(-) Mo:T cell cocultures. Engagement of CCR3 and CCR4 by CCL24 and CCL2, respectively, along with stimulation via CD3/CD28, renders T cells highly permissive for productive HIV infection. Moreover, HIV replicates preferentially in CCR3(+) and CCR4(+) T cells. These findings reveal a new pathway of T cell costimulation for increased susceptibility to HIV infection via engagement of CCR3 and CCR4 by chemokines constitutively produced by CD16(+) Mo/Mphi. Thus, expansion of CD16(+) Mo in peripheral blood of HIV-infected patients and their subsequent recruitment into tissues may contribute to chronic immune activation and establishment of viral reservoirs in resting T cells.     

The restricted expression of granzyme M in human lymphocytes

We have analyzed the expression of human granzyme M (Gzm M) in various human leukocyte subsets using the specific mAb 4H10. Using FACS and Western blotting analysis we compared the expression of Gzm M with that of other granzymes (Gzm A and Gzm B) and the lytic protein perforin. Human Gzm M was constitutively highly expressed in NK cells as was perforin and Gzm A. Surprisingly, freshly isolated NK cells had very low (sometimes undetectable) levels of Gzm B. In contrast to Gzm B and perforin, Gzm M was not detected in highly purified CD4(+) and CD8(+) T cells either constitutively or after short term activation in vitro. However, low levels of Gzm M were observed in some T cell clones on prolonged passage in vitro. Gzm M was not detected in highly purified neutrophils, monocytes, or tumor cells of the myelomonocytic lineage. Examination of minor T cell subsets from human peripheral blood showed detectable Gzm M in CD3(+), CD56(+) T cells and gammadelta T cells. A histological staining procedure was developed that demonstrated a granular staining pattern for Gzm M and a cellular distribution similar to that observed by Western blotting. These data indicate that the expression of Gzm M does not always correlate with the lytic activity of cytotoxic cells. However, expression of Gzm M in NK cells, CD3(+), CD56(+) T cells, and gammadelta T cells suggests that this enzyme may play some role in innate immune responses.     

CD4(+) T cells are required for the development of cytotoxic CD8(+) T cells during Mycobacterium tuberculosis infection.

The control of acute and chronic Mycobacterium tuberculosis infection is dependent on CD4(+) T cells. In a variety of systems CD8(+) T cell effector responses are dependent on CD4(+) T cell help. The development of CD8(+) T cell-mediated immune responses in the absence of CD4(+) T cells was investigated in a murine model of acute tuberculosis. In vitro and in vivo, priming of mycobacteria-specific CD8(+) T cells was unaffected by the absence of CD4(+) T cells. Infiltration of CD8(+) T cells into infected lungs of CD4(-/-) or wild-type mice was similar. IFN-gamma production by lung CD8(+) T cells in CD4(-/-) and wild-type mice was also comparable, suggesting that emergence of IFN-gamma-producing mycobacteria-specific CD8(+) T cells in the lungs was independent of CD4(+) T cell help. In contrast, cytotoxic activity of CD8(+) T cells from lungs of M. tuberculosis-infected mice was impaired in CD4(-/-) mice. Expression of mRNA for IL-2 and IL-15, cytokines critical for the development of cytotoxic effector cells, was diminished in the lungs of M. tuberculosis-infected CD4(-/-) mice. As tuberculosis is frequently associated with HIV infection and a subsequent loss of CD4(+) T cells, understanding the interaction between CD4(+) and CD8(+) T cell subsets during the immune response to M. tuberculosis is imperative for the design of successful vaccination strategies.     

Prognostic value of the immunological phenomena and relationship with clinicopathological characteristics of the tumor--the expression of the early CD69+, CD71+ and the late CD25+, CD26+, HLA/DR+ activation markers on T CD4+ and CD8+ lymphocytes in squamous cell laryngeal carcinoma. Part II

One of the most important challenges in contemporary oncology is to find objective biomarkers of tumor aggressiveness, which help to identify more invasive phenotypes of the carcinoma. The purpose of this study was to investigate the relationships between the early and the late activation markers expression on T CD4(+) and CD8(+) cells subpopulations and certain clinicopathological characteristics of the neoplastic infiltration in order to determine their role as biomarkers for tumor behavior in squamous cell laryngeal carcinoma. Analysis of the early (CD69(+), CD71(+)) and the late activation antigens (CD25(+) (high), CD26(+), HLA/DR(+)) expression on T CD4+ and CD8(+) lymphocytes by cytofluorymetry in 55 patients treated for squamous cell laryngeal carcinoma was performed. Clinicomorphological analysis on the basis of TNM criteria and tumor front grading, which included tumor-related features and adjacent stroma-related characteristics of the peripheral edge of infiltration was carried out. The relationships between the activation markers expression and parameters of tumor aggressiveness were investigated. Our work revealed statistically significant differences in the expression of the studied activation markers on T cells with regard to certain clinicomorphological features. The expressions of CD69(+) and CD71(+) antigens on T CD3(+)CD4(+) and CD3(+)CD8(+) cells as well as CD4(+)HLA/DR(+) markers were higher for pT3 and pT4 tumors, in comparison with pT2 carcinomas. Moreover, tumors with the smallest number of TFG points were characterized by significantly lower values of the average expression of CD3(+)CD69(+) and CD3(+)CD71(+) as well as CD4(+)HLA/DR(+) markers on T lymphocytes. In addition, more aggressive and deeply infiltrating laryngeal carcinomas were most often characterized by significantly higher values of the average expression of CD69(+) and CD71(+) antigens on CD8(+) as well as HLA/DR(+) markers on CD4(+). Our study confirmed the implication of the early and the late activation antigens expression on CD4(+) and CD8(+) T lymphocytes in clinicomorphological parameters of the tumor, especially TFG total score and depth of invasion, and their importance as indicators of the invasive phenotype of laryngeal carcinoma.     

Adrenalectomy in mice does not prevent loss of intestinal lymphocytes after exercise.

Exhaustive exercise is associated with an increase in circulating glucocorticoids (GCs), lymphocyte apoptosis, and a reduction in intestinal lymphocyte number. The present study examined the role of GCs on the numerical changes seen in intestinal lymphocytes after exercise. Female C57BL/6 mice were bilaterally adrenalectomized (ADX; n = 18) or given sham surgery (Sham; n = 18) and assigned to one of three exercise conditions: treadmill running (28 m/min, 90 min, 2 degrees slope) and killed immediately or after 24 h recovery, or not exercised and killed immediately after 90-min exposure to the treadmill environment. Lymphocytes were isolated from the intestines with CD45(+) cells collected by positive selection using magnetic bead separation columns, and lymphocyte subpopulations were analyzed by flow cytometry for CD45(+), CD3alphabeta(+), CD3gammadelta(+), CD8beta(+), CD8alpha(+), CD4(+), and NK(+) phenotypic markers. ADX mice had significantly more intestinal CD45(+) leukocytes (P < 0.05) and CD3alphabeta(+) (P < 0.05), CD3gammadelta(+) (P < 0.01), CD8alpha(+) (P < 0.001), and NK(+) (P < 0.05) intestinal lymphocytes than Sham mice. There was a significant effect of exercise condition on total intestinal CD45(+) leukocytes (P < 0.01) and CD3alphabeta(+) (P < 0.05), CD8alpha(+) (P < 0.001), and CD4(+) (P < 0.05) intestinal lymphocytes, with fewer cells at 24 h postexercise compared with the other treatment conditions. There were no surgical x exercise interaction effects on the CD3 and CD8 phenotype numbers. Plasma corticosterone was virtually nil in ADX mice regardless of exercise condition but was significantly elevated in Sham mice immediately postexercise (P < 0.001). The data indicate that ADX does not prevent the loss of lymphocytes from the intestinal mucosa 24 h after strenuous exercise and GCs are not directly causal in the leukopenia of exercise.