L3T4(CD4)-, Lyt-2(CD8)- and Mac-1(CD11b)-phenotypic leukocytes in murine cryptococcal meningoencephalitis

An immunohistological study of L3T4(CD4)+ and LYT-2(CD8)+ lymphocytes, Mac-1(CD11b)+ monocytes and granulocytes in experimental murine cryptococcal meningoencephalitis was conducted. To assess the concomitant inflammatory reaction in an extracerebral site, livers were examined in parallel. Mice were infected i.v. withCryptococcus neoformans, group A/D, and organs were examined immunohistologically for CD4-, CD8- and monocyteand granulocyte-specific CD11b-phenotypic leukocytes over a period of 60 days. Intracerebrally, agglomerations of cryptococci formed pseudocysts that were surrounded by CD4+ and CD8+ lymphocytes at the end of the second week post-infection, followed by the invasion of monocytes and granulocytes into the lesions. After the fourth week post-infection, most of the invaded lesions were transformed into glious scars. Meningitis was usually marked and showed a homogenous distribution of CD4-, CD8- and CD11b-phenotypic cells, with a predominance of monocytes and CD4+ lymphocytes. Inflammatory infiltrates in the liver were found already 4 days post-infection. CD4+ lymphocytes and monocytes were distributed homogenously in the infiltrates, with a lower number of CD8+ lymphocytes being located rather in the periphery of the infiltrates. Comparing leukocyte kinetics in brain and liver, an important observation was the delayed immigration of immune cells at the intracerebral cryptococcal lesions as compared with the liver, and the different migration patterns of T-lymphocyte subgroups and macrophages. These results suggest that there are differential leukocyte migration patterns in the liver and brain following disseminated cryptococcosis. The immunological aspects of the observed leukocyte kinetics are discussed.     

Human epidermal cells from ultraviolet light-exposed skin preferentially activate autoreactive CD4+2H4+ suppressor-inducer lymphocytes and CD8+ suppressor/cytotoxic lymphocytes

In vivo exposure of human epidermis to UV abrogates the function of T6+DR+ Langerhans cells and induces the appearance of Ag-presenting T6-DR+ OKM5+ cells in the epidermis. Since UV exposure of murine skin results in Ts lymphocyte activation, we investigated the capacity of human epidermal cells (EC) harvested 3 days after in vivo UV exposure to activate regulatory and effector autologous T lymphocyte subsets. T lymphocytes were separated into CD8+ suppressor/cytotoxic lymphocytes and CD4+ helper/inducer lymphocytes by C lysis and panning. The CD4+ subset was further divided by using the 2H4 mAB to obtain CD4+2H4+ lymphocytes (inducers of TS lymphocytes) and CD4+2H4- lymphocytes (inducers of B cell Ig production and inducers of cytotoxic T cells). Unirradiated suction blister-derived EC from control skin (C-EC) and from skin exposed in vivo to UV (UV-EC) were cultured with purified autologous T lymphocyte subsets in the absence of added Ag. The resultant T lymphocyte proliferation was detected by [3H]thymidine uptake. UV-EC were highly effective in the stimulation of CD4+ lymphocytes, whereas C-EC were poor stimulators. The stimulator effect of UV-EC was abrogated after depletion of DR+ UV-EC. When CD4+ lymphocytes were fractionated, UV-EC consistently demonstrated enhanced ability to stimulate suppressor-inducer CD4+2H4+ lymphocytes relative to C-EC. Although less responsive than CD4+2H4+ lymphocytes, CD4+2H4- lymphocytes also demonstrated greater proliferation to UV-EC than to C-EC. Neither UV-EC nor C-EC were able to activate CD8+ lymphocytes devoid of CD4+ lymphocytes. However, after addition of rIL-2 at concentrations that allow binding only to the high affinity IL-2R on T lymphocytes, UV-EC induced vigorous proliferation of CD8+ lymphocytes, whereas C-EC induced only background levels of proliferation. C lysis of leukocytes resident within UV-EC resulted in 66 to 70% reduction of CD8+ lymphocyte proliferation. In conclusion, UV-EC may activate CD8+ lymphocytes by at least two pathways: (1) UV-EC activation of CD4+2H4+ lymphocytes may induce differentiation/proliferation of CD8+ suppressor cells and (2) UV-EC activation of CD4+ cells may induce IL-2 production, that, in combination with UV-induced epidermal leukocytes, stimulates CD8+ cells.     

Regulation of beta2-adrenergic receptors on CD4 and CD8 positive lymphocytes by cytokines in vitro

Increasing evidence points to a close relationship between the autonomic nervous system and the immune system. To further investigate mechanisms regulating beta2-adrenergic receptor (beta2R) expression in lymphocytes, the influence of cytokines on the density of beta2R on purified CD4+ and CD8+ lymphocytes was determined in vitro. beta2R were determined by means of a radioligand binding assay with (125I)iodocyanopindolol. CD4+ and CD8+ lymphocytes were incubated with catecholamines, interleukin 1beta (IL-1beta) and interleukin 2 (IL-2) for 6-72 h. The results demonstrate declining beta2R numbers on CD4+ and CD8+ lymphocytes in vitro augmented by epinephrine. IL-1beta has no effect on beta2R expression compared to medium. However, incubation with IL-2 resulted in an up-regulation of beta2R on CD8+ lymphocytes. Thus, the study demonstrates a differential regulation of beta2R on T-lymphocyte subpopulations with CD8+ lymphocytes being more susceptible to mechanisms of beta2R modulation than CD4+ lymphocytes. The findings further strengthen the concept of a close interplay between the autonomic nervous system and the immune system.     

CD4-deficient mice have reduced levels of memory cytotoxic T lymphocytes after immunization and show diminished resistance to subsequent virus challenge.

Although primary antiviral CD8+ cytotoxic T lymphocytes (CTL) can be induced in mice depleted of CD4+ T cells, the role of CD4+ T lymphocytes in the generation and maintenance of antiviral memory CTL is uncertain. This question, and the consequences upon vaccine-mediated protection, were investigated in transgenic CD4 knockout (CD4ko) mice, which lack CD4+ T lymphocytes. Infection of immunocompetent C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV), or with recombinant vaccinia viruses bearing appropriate LCMV sequences, induces long-lasting protective immunity, mediated mainly by antiviral CD8+ CTL. Here we report two important findings. First, LCMV-specific CD8+ memory CTL are maintained at considerably lower levels in CD4ko mice than in normal C57BL/6J mice; we demonstrate a reduction in precursor CTL evident as soon as 30 days postimmunization and declining, by day 120, to levels 1 to 2 log units below those in normal mice. Thus, CD4+ T cells appear to be important to the generation and maintenance of their CD8+ counterparts. Second, this reduction has an important biological consequence; compared with immunocompetent mice, CD4ko mice immunized with vaccinia virus recombinants expressing nucleoprotein or glycoprotein of LCMV are less effectively protected from subsequent LCMV challenge. Thus, this study underscores the potential importance of CD4+ T lymphocytes in generation of appropriate levels of CD(8+)-cell-mediated immunoprotective memory and has implications for vaccine efficacy in individuals with immune defects in which CD4 levels may be reduced, such as AIDS.     

Characterization of the CD4+ and CD8+ tumor infiltrating lymphocytes propagated with bispecific monoclonal antibodies

To study the CD4+ and CD8+ tumor infiltrating lymphocytes (TIL) in the antitumor response, we propagated these subsets directly from tumor tissues with anti-CD3:anti-CD8 (CD3,8) and anti-CD3:anti-CD4 (CD3,4) bispecific mAb (BSMAB). CD3,8 BSMAB cause selective cytolysis of CD8+ lymphocytes by bridging the CD8 molecules of target lymphocytes to the CD3 molecular complex of cytolytic T lymphocytes with concurrent activation and proliferation of residual CD3+CD4+ T lymphocytes. Similarly, CD3,4 BSMAB cause selective lysis of CD4+ lymphocytes whereas concurrently activating the residual CD3+CD8+ T cells. Small tumor fragments from four malignant melanoma and three renal cell carcinoma patients were cultured in medium containing CD3,8 + IL-2, CD3,4 + IL-2, or IL-2 alone. CD3,8 led to selective propagation of the CD4+ TIL whereas CD3,4 led to selective propagation of the CD8+ TIL from each of the tumors. The phenotypes of the TIL subset cultures were generally stable when assayed over a 1 to 3 months period and after further expansion with anti-CD3 mAb or lectins. Specific 51Cr release of labeled target cells that were bridged to the CD3 molecular complexes of TIL suggested that both CD4+ and CD8+ TIL cultures have the capacity of mediating cytolysis via their Ti/CD3 TCR complexes. In addition, both CD4+ and CD8+ TIL cultures from most patients caused substantial (greater than 20%) lysis of the NK-sensitive K562 cell line. The majority of CD4+ but not CD8+ TIL cultures also produced substantial lysis of the NK-resistant Daudi cell line. Lysis of the autologous tumor by the TIL subsets was assessed in two patients with malignant melanoma. The CD8+ TIL from one tumor demonstrated cytotoxic activity against the autologous tumor but negligible lysis of allogeneic melanoma targets. In conclusion, immunocompetent CD4+ and CD8+ TIL subsets can be isolated and expanded directly from small tumor fragments of malignant melanoma and renal cell carcinoma using BSMAB. The resultant TIL subsets can be further expanded for detailed studies or for adoptive immunotherapy.     

IFN-gamma and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells

Host defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-gamma by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-gamma gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-gamma message in animals treated with the IFN-gamma gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer.     

Infection of the CD45RA+ (naive) subset of peripheral CD8+ lymphocytes by human immunodeficiency virus type 1 in vivo

To investigate the mechanism and functional significance of infection of CD8+ lymphocytes by human immunodeficiency virus type 1 (HIV-1) in vivo, we determined frequencies of infection, proviral conformation, and genetic relationships between HIV-1 variants infecting naive (CD45RA+) and memory (CD45RO+) peripheral blood CD4+ and CD8+ lymphocytes. Infection of CD3+ CD8+ CD45RA+ cells was detected in 9 of 16 study subjects at frequencies ranging from 30 to 1,400 proviral copies/10(6) cells, more frequently than CD3+ CD8+ lymphocytes expressing the RO isoform of CD45 (n = 2, 70 and 260 copies /10(6) cells). In agreement with previous studies, there was no evidence for a similar preferential infection of CD4+ naive lymphocytes. Proviral sequences in both CD4+ and CD8+ lymphocyte subsets were complete, as assessed by quantitation using primers from the long terminal repeat region spanning the tRNA primer binding site. In six of the seven study subjects investigated, variants infecting CD8+ lymphocytes were partially or completely genetically distinct in the V3 region from those recovered from CD4+ lymphocytes and showed a greater degree of compartmentalization than observed between naive and memory subsets of CD4+ lymphocytes. In two study subjects, arginine substitutions at position 306, associated with use of the chemokine coreceptor CXCR4, were preferentially found in CD4 lymphocytes. These population differences may have originated through different times of infection rather than necessarily indicating a difference in their biological properties. The preferential distribution of HIV-1 in naive CD8+ lymphocytes indeed suggests that infection occurred early in T-lymphocyte ontogeny, such as during maturation in the thymus. Destruction of cells destined to become CD8+ lymphocytes may be a major factor in the decline in CD8+ lymphocyte frequencies and function on disease progression and may contribute directly to the observed immunodeficiency in AIDS.     

CD4+CD8- thymocytes from neonatal mice induce IgM production in SCID mice.

Defective recombination of both the TCR and Ig genes results in the absence of mature lymphocytes in mice with the scid mutation. We have shown previously that the transfer of neonatal, but not adult, thymocytes results in high levels of Ig production in 100% of C.B-17-scid (SCID) mice, in contrast to the 10 to 25% of SCID mice spontaneously producing low levels of oligoclonal Ig. In this report we demonstrate that neonatal CD4+8- thymocytes were able to induce this response; the CD4+8+ and CD4-8+ subpopulations were totally inactive and CD4-8- T cells had only limited activity several weeks after transfer. The stimulation of IgM production in SCID mice was detectable by 1 wk posttransfer of CD4+8- thymocytes or splenic T cells, and could be achieved with as few as 300 cells. The ability of neonatal CD4+8- thymocytes to induce Ig diminished gradually to insignificant levels at 3 wk postbirth; this loss of function was not associated with differential survival of neonatal T cells. Neonatal CD4+8- thymocytes from C.B-17 and other H-2d strains rescued Ig production, whereas cells from H-2b, H-2a, and H-2k strains were much less effective. These results suggest that a CD4+8- subpopulation found in both neonatal thymus and peripheral lymphoid tissues is able to induce the expansion or differentiation of the small numbers of functional B lymphocytes in SCID mice, and that the inducing T cell disappears shortly after birth, perhaps during the acquisition of self-tolerance.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

High levels of human immunodeficiency virus infection of CD8 lymphocytes expressing CD4 in vivo

Human immunodeficiency virus (HIV)-infected CD8 lymphocytes have been reported in vivo, but the mechanism of infection remains unclear. Experiments using the thy/hu mouse model support export of intrathymically infected CD8 precursors, while recent in vitro data suggest that mature CD8 lymphocytes upregulate CD4 upon activation (generating a CD8bright CD4dim phenotype) and are susceptible to HIV infection. To determine whether these mechanisms operate in vivo and to assess their relative importance in the generation of circulating HIV-infected CD8 lymphocytes, we quantified HIV long terminal repeat (LTR) DNA in CD8+ CD4- and CD8bright CD4dim lymphocytes isolated from HIV-infected individuals by fluorescence-activated cell sorting. HIV infection of CD8 lymphocytes was demonstrated in 17 of 19 subjects, with a significant inverse relationship between level of infection and CD4 lymphocyte count (R = -0.73; P < 0.001). The level of HIV infection of CD8bright CD4dim lymphocytes was significantly higher (median, 1,730 HIV LTR copies/10(6) cells; n = 9) than that of CD8+ CD4- lymphocytes (undetectable in seven of nine individuals; P < 0.01) and approached that of CD4 lymphocytes from the same individuals (median, 3,660 HIV LTR copies/10(6) cells). CD8bright CD4dim lymphocytes represented 0.8 to 3.3% of total CD8 lymphocytes and were most prevalent in the memory subset. Thus, HIV-infected CD8 lymphocytes commonly circulate in HIV-infected individuals and are generated through infection of activated CD8 lymphocytes rather than through export of intrathymically infected precursors. The high level of infection of CD8bright CD4dim lymphocytes could have a direct role in the decline in CD8 lymphocyte function that accompanies HIV disease progression.     

The role of CD4+ cells in sustaining lymphocyte proliferation during lymphocytic choriomeningitis virus infection.

The murine immune response to lymphocytic choriomeningitis virus (LCMV) infection involves the activation of CD8+, class I MHC-restricted and virus-specific CTL. At times coinciding with CTL activation, high levels of IL-2 gene expression and production occur, the IL-2R is expressed, and T cell blastogenesis and proliferation are induced. We have previously found that, although both CD4+ and CD8+ T cell subsets transcribe IL-2, the CD4+ subset appears to be the major producer of IL-2 whereas the CD8+ subset appears to be the major proliferating population when the subsets are separated after activation in vivo. The studies presented here were undertaken to examine the contribution made by the CD4+ subset to lymphocyte proliferation in vivo. Responses to LCMV infection were examined in intact mice and in mice depleted of CD4+ or CD8+ subsets by antibody treatments in vivo. Protocols were such that in vivo treatments with anti-CD4 or anti-CD8 depleted the respective subset by greater than 90%. In situ hybridizations demonstrated that the IL-2 gene was expressed in non-B lymphocytes isolated from either CD4+ cell-depleted or CD8+ cell-depleted mice on day 7 post-infection with LCMV. When placed in culture, however, cells from CD8+ cell-depleted mice produced significantly higher levels of detectable IL-2 than did cells isolated from CD4+ cell-depleted mice on day 7 post-infection. IL-2 was apparently produced in vivo in mice depleted of either CD4+ or CD8+ cells, as expression of the gene for the p55 chain of the IL-2R, IL-2 responsiveness, and lymphocyte proliferation were observed with cells isolated from both sets of mice. Lymphocyte proliferation was shown to be sustained in mice depleted of CD4+ cells in vivo by three criteria: 1) non-B lymphocytes isolated from infected mice depleted of CD4+ cells underwent more DNA synthesis than did those isolated from uninfected mice or from infected mice depleted of CD8+ cells; 2) leukocyte yields were expanded during infection of CD4+ cell-depleted mice; and 3) CD8+ cell numbers were increased during infection of CD4+ cell-depleted mice. The majority of non-B lymphocytes having the characteristics of blast lymphocytes was recovered in the CD8+ populations isolated from infected CD4+ cell-depleted mice. These findings suggest that the requirement for the CD4+ subset to sustain CD8+ lymphocyte proliferation in vivo is limited, and that CD4+ and CD8+ cell types can function independently in many aspects of their responses to viral infections.     

Antigen-specific functions of a CD4+ subset of human T lymphocytes with granular morphology

We previously showed that a small proportion of the CD4+ human lymphocytes express the C3bi receptor (CD11). In the present investigation these cells were found to have the homogeneous morphology of granular lymphocytes. Between 70 and 80% of the cells reacted with antibody Leu-7, but they did not express Fc receptors and had no spontaneous cytotoxic activity against the NK-sensitive cell line K562. The CD11+/CD4+ cells uniformly expressed CD3 and could be induced to express IL 2 receptors by activation with PHA or anti-CD3 antibody. Compared with unfractionated T cells, however, CD11+/CD4+ granular lymphocytes had a reduced ability to produce IL 2 after stimulation with PHA or anti-CD3 antibody. Nevertheless, the CD11+/CD4+ subset generated specific allocytotoxicity after stimulation with allogeneic cells and culture in IL 2. Furthermore, the CD11+/CD4+ cells also proliferated after stimulation with tetanus toxoid and PPD. Thus some cells of the CD11+/CD4+ subset are capable of antigen-specific responses. These results indicate that certain CD11+ cells with granular morphology are mature T lymphocytes.     

The subpopulation structure of human T-lymphocytes studied by using a monoclonal antibody (MCA) to CD27]

The obtained murine mAb LT27 (IgG2a) assigned to the cluster of differentiation CD27 was used to study the distribution of antigen CD27 among human lymphocytes scbpopulations in normal state and immunopathology. In normal donors the antigen CD27 was found to be expressed most frequently on CD4+ cells (90 +/- 8% of which coexpressed antigen CD27) and to the lesser extent on- CD8+ cells (only 77 +/- 28% of CD8+ cells carried antigen CD27). 79 +/- 12% of double negative lymphocytes (CD3+CD4-CD8-) expressed antigen CD27. In patients with hypogammaglobulinemia the proportion of CD4+CD27+/CD4+ and CD8+CD27+/CD8+ was significantly reduced to 80 +/- 11% (p < 0.01) and 45 +/- 19% (p < 0.001), respectively. The ratio CD4+CD27+/CD4+ varied insignificantly with the increase of CD4+ population, but the increase of the CD8+ population was accompanied by the definite tendency to a decrease of the ratio CD8+CD27+/CD8+. The distribution of CD27 antigen inside CD4+ and CD8+ subpopulations was found to be different from the distribution of CD29 and CD45RA antigens.     

The CD160+ CD8high cytotoxic T cell subset correlates with response to HAART in HIV-1+ patients

We investigated the circulating cytotoxic CD160+ CD8(high) subset in correlation to antiviral immunity and response to highly active antiretroviral therapy (HAART) in HIV+ subjects. The study included 45 treatment-naive patients receiving HAART for 18 months, retrospectively defined as good (n=29) and transient (n=16) responders. HIV-specific CD8 T lymphocyte levels were measured by IFNgamma production in response to p17 Gag, in the presence of immobilized anti-CD160 mAb. We report a significantly increased baseline level of CD160+ CD8(high) subset in good therapy responders. CD160+ CD8(high) subset correlates with CD4+ T cell count, immune activation, and viral load. CD160+ CD8(high) lymphocytes contain a high amount of Granzyme B and include virus-specific T lymphocytes in HIV-1+ subjects. Co-stimulation through CD160 molecules enhances IFNgamma production in response to p17 Gag. Therefore, the CD160+ CD8(high) subset may be useful for monitoring of virus-specific cellular immunity and predicting response to antiretroviral therapy in chronic HIV-1 infection.     

Radiosensitivity of CD4 or CD8 positive human T-lymphocytes by an in vitro colony formation assay

The recent development of an in vitro lymphocyte colony assay makes it possible to examine variations in the radiosensitivity of humans using peripheral blood lymphocytes (PBL) instead of the skin fibroblast assay. Our recent study (M. Hakoda et al., Mutat. Res. 197, 161-169, 1988) showed that most of the colonies consisted of lymphocytes bearing CD4 or CD8 antigens. Since the fraction of CD4+ and CD8+ cells in PBL differs among individuals, we suspected that individual radiosensitivity might be biased by the different subset frequencies if the dose-survival curves of the CD4+ and CD8+ cells were different from each other. In the present study, CD4+ (helper/inducer T) and CD8+ (suppressor/cytotoxic T) lymphocytes were isolated from PBL and their dose-survival curves were determined. The results showed that the D10 (dose required to reduce the surviving fraction to 10%) was similar for these two types of cells [3.13 +/- 0.10 Gy (mean +/- SD) for CD4+, 3.34 +/- 0.50 Gy for CD8+ and 3.14 +/- 0.17 Gy for the unsorted cells], supporting the use of the whole PBL population for the screening of individuals with altered radiosensitivity.     

Simian immunodeficiency virus infection of CD8+ lymphocytes in vivo.

To determine the lymphoid target cells of simian immunodeficiency virus (SIV) in vivo, peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) were positively selected (>97% purity) for surface expression of CD4, CD8, or CD20 and then analyzed for SIV provirus using semiquantitative DNA amplification. We found provirus in CD4+ and CD8+ lymphocytes but none in CD20+ lymphocytes. During acute SIV infection (< or = 214 days postinoculation), the percentage of PBL and LNL CD4+ cells containing proviral DNA ranged from 0.2 to 20% and from 0.2 to 2%, respectively. Proviral burden in the CD8+ population of either PBL or LNL ranged from 0.01 to 0.2%. Virus isolation by cocultivation was positive for both CD4+ and CD8+ purified populations. No difference in proviral burden was observed between PBL and LNL subsets during acute SIV infection. Up to 19.4% of positively selected CD8+ cells also expressed CD4, and thus the provirus may reside within a dual-positive population. This dual-positive population may represent activated lymphocytes that are particularly susceptible to infection and may provide an opportunity for virus entry into the CD8+ CD4- lymphocytes in vivo.     

Simultaneous increased expression of E-rosette receptor (CD2, T11) and T cell growth factor receptor on human T lymphocytes during activation

The E-rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3-Ti complex (anti-Leu-4) caused the expression of CD2 to increase 10- to 20-fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti-Tac) and anti-CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin-D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cells, respectively. Although virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8- subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20-30 hr after activation, the expression of CD4 or CD8 was initially decreased 10-50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therefore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.     

Effector activity of decidual CD8+ T lymphocytes in early human pregnancy

Although CD8+ T lymphocytes are present in human decidua throughout pregnancy, albeit as a minor population in early pregnancy, their role in normal pregnancy is largely unknown. The present study aimed to characterize their effector phenotype, including cytolytic activity, cytokine profile, and capacity to affect placental invasion. CD8+ lymphocytes were positively selected from normal early pregnancy decidua (7-14 wks gestational age). Decidual CD8+ T lymphocytes were studied using standard and redirected chromium release assays to investigate natural killer cell-sensitive cytotoxicity and cytotoxicity that requires T-cell receptor signal transduction respectively, multiplex cytokine analysis to analyze cytokine production, and a placental explant invasion model to assess the effect of soluble products of decidual CD8+ T lymphocytes on trophoblast invasion. Decidual CD8+ T lymphocytes exhibited cytolytic ability against P815 target cells (mean % Specific Chromium Release at effector:target ratio of 32:1 [SCR(32)] of 32.7 +/- 5.8) and against K562 target cells (mean SCR(32) of 20.3 +/- 0.5). Phytohemagglutinin-P (PHA-P)-stimulated decidual CD8(+) T lymphocytes produced high levels of both interferon gamma and interleukin (IL) 8, and low levels of granulocyte-macrophage colony-stimulating factor (CSF2), IL1B, IL2, IL6, IL10, IL12, and tumor necrosis factor; these did not vary with gestational age. IL4 was undetectable. Decidual CD8+ T lymphocyte supernatants increased the capacity of extravillous trophoblast cells to invade through Matrigel compared with the PHA-P control. These findings suggest that decidual CD8+ T cells can display cytolytic activity, do not evoke a predominant local intrauterine Th2 type cytokine environment, and may act to regulate invasion of extravillous trophoblast cells into the uterus, a crucial process for normal uteroplacental development.