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.     

CD11 molecule defines two types of suppressor cells within the T8+ population

It has been shown that T8+ cells are comprised of functionally heterogeneous subpopulations such as suppressor, cytotoxic, and NK cells. In this report, we attempted to delineate the functional heterogeneity of T8 cells defined by anti-CD11 antibody (anti-Mol). Although allospecific cytotoxic activity was restricted to the T8+Mol- subset, suppression of PWM IgG synthesis could be elicited in both the T8+Mol+ and the T8+Mol- subset of cells. However, the mechanism of suppression was different in these two subsets. Suppression by the T8+Mol- subset of cells required the interaction with the T4+2H4+ suppressor inducer cells, whereas the T8+Mol+ subset of cells could suppress in the absence of the suppressor inducer cells. Moreover, recombinant interleukin-2 alone could augment this suppression by the T8+Mol+ subset, but did not induce suppression by the T8+Mol- subset. In contrast, NK and LAK activity was exclusively found in the T8+Mol+ subset of cells but not in the T8+Mol- subset of cells. These results suggest that the CD11 molecule is useful for distinguishing novel subsets of T8 cells.     

Subpopulations of CD8+ cytotoxic T cell precursors collaborate in the absence of conventional CD4+ helper T cells.

Four different subpopulations (Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi) of CD8+ T cells were arbitrarily defined on the basis of differential expression of Ly6C Ag. By combining the processes of electronic cell sorting and automated cell deposition, small numbers of respective CD8+ T cell subpopulations were directly deposited into tissue culture wells in which mitogen-stimulated responses were studied. Anti-CD3-stimulated proliferation and IL-2 production were the strongest by Ly6Cneg/Ly6Clow T cells, moderate for Ly6Cint T cells, and highly deficient for Ly6Chi T cells. The level of IL-2 production for Ly6Cneg CD8+ T cells was comparable to that of conventional CD4+ Th cells. Allogeneic stimulator cells elicited a strong cytotoxic response by Ly6Cneg + low but not Ly6Chi CD8+ T cells in the absence of added lymphokines. When IL-2 was supplied in excess, anti-CD3 induced comparable levels of cell proliferation and cytotoxic activity in Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi CD8+ T cells whereas alloantigen stimulated an approximate fivefold higher cytotoxic response by Ly6Chi than Ly6Cneg + low CD8+ T cells. Stimulation of co-cultures of B10 (CD8b) Ly6Cneg + low and congenic B10.CD8a Ly6Chi CD8+ T cells in the absence of added lymphokines, followed by selective elimination of activated CD8.1+ (CD8.2+) T cells by anti-CD8.1 (anti-CD8.2) + C treatment, allowed the demonstration that help provided by Ly6Cneg + low T cells can be effectively used by both Ly6Cneg + low and Ly6Chi T cells in anti-CD3 and alloantigen induced proliferative and cytotoxic responses, respectively.     

Antigen presentation by Toxoplasma gondii-infected cells to CD4+ proliferative T cells and CD8+ cytotoxic cells

Cytotoxic cells specific for Toxoplasma gondii-infected cells were detected in the peripheral blood leukocytes from a patient with acute toxoplasmosis. The cytotoxicity was mediated by CD5+, CD4-, CD8+ cells. The cytotoxic T cells lysed Toxoplasma-infected target cells with HLA class I restriction. Two types of T cell clones were established from peripheral blood leukocytes of a patient with chronic toxoplasmosis; one was a CD5+, CD4-, CD8+ cytotoxic cell specific for Toxoplasma-infected cells, and the other was a CD5+, CD4+, CD8- proliferative cell that responded to Toxoplasma antigen. Toxoplasma-infected cell-specific cytotoxic cloned T cells recognize the infected target cells in the context of the HLA class I molecules, and the CD8 molecule was involved in the cytotoxicity. Toxoplasma antigen-specific proliferative cloned T cells were stimulated by Toxoplasma antigen-pulsed or Toxoplasma-infected cells in conjunction with HLA-DR molecule on the target cells. Thus, antigen presentation by Toxoplasma-infected cells for activation of both cytotoxic and proliferative T cells has been demonstrated.     

Early development of CD8-negative and CD8-positive MHC-unrestricted cytotoxic cells induced by alloantigen inoculation in mice

Peritoneal cells (PC) in C57B1/6 (B6, H-2b) mice receiving an intraperitoneal (i.p.) injection of allogeneic BALB/c (H-2d) spleen cells demonstrated potent cytotoxic activity against syngeneic, xenogeneic, third-party allogeneic tumors as well as H-2d derived tumors. Maximum cytotoxic activity against various tumors other than H-2d derived tumor, B16 (H-2b) was elicited on day 3 post allosensitization and decreased drastically thereafter, whereas cytotoxic activity against P815 (H-2d) peaked 3 days after the inoculation and maintained the peak activity thereafter. Surface phenotype of PC responsible for the cytotoxic activity against B16 was Thy-1+/-, Lyt-2-, L3T4-, asialo GM1 (AGM1)+, and that of PC against P815 was Thy-1+, Lyt-2+ (or Lyt-2+/-), L3T4-, AGM1+. These phenotypes showed similar phenotypes to the counterparts against B16 and against P815 in spleen cells induced by intravenous inoculation of alloantigen. When mice were pretreated i.p. with anti-AGM1 antibody before the allosensitization, anti-P815 cytotoxic-activity in PC was completely diminished. Similar activity in spleen, however, was enhanced by i.v. treatment with the antibody before the i.v. inoculation of alloantigen. The data clearly demonstrate that in vivo inoculation of B6 mice with normal allogeneic cells induces "NK-like" CD8- cytotoxic cells and "anomalous" CD8+ cytotoxic cells in PC.     

CD4-8- dendritic cells prime CD4+ T regulatory 1 cells to suppress antitumor immunity

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of OVA-presenting CD4-8-, CD4+8-, or CD4-8+ DCs (OVA-pulsed DC (DC(OVA))) in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD4-8- DCs did so only weakly. Both CD4+8- and CD4-8+ DC(OVA) induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ CTL-mediated antitumor immunity, whereas CD4-8- DC(OVA), which were less mature and secreted substantial TGF-beta upon coculture with TCR-transgenic OT II CD4+ T cells, induced the development of IL-10-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD4-8- DC(OVA) and IL-10-/- OT II CD4+ T cells, into CD4-8+ DC(OVA)-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD4-8- DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8- and CD4-8+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity.     

A previously unrecognized large fraction of cytotoxic lymphocyte precursors is present in CD4+ human peripheral blood T cells

We describe a limiting dilution (LD) culture system in which cell sorter-purified CD4+ (and CD8+) peripheral blood T cells are cocultured with irradiated, anti-CD3 mab-producing OKT3 hybridoma cells. Under these conditions, one out of 2-3 CD4+ (and CD8+) T cells is induced to clonal proliferation. In striking contrast to previously described LD culture systems, every growing CD4+ cell clone displayed cytotoxic activity when tested in a lectin-facilitated 51Cr release assay against P815 target cells. This contrasts with the development of cytotoxic CD4+ T cells in alloantigen-stimulated LD cultures, where only one out of 15-20 proliferating CD4+ cells killed P815 in the presence of PHA, and one out of 300-500 proliferating CD4+ cells displayed alloantigen-specific cytotoxic activity. Furthermore, we have established antigen-specific proliferating CD4+ T cell clones which do not exert antigen-specific cytotoxicity but can be cytotoxic when crosslinked to target cells via lectin or monoclonal antibody (anti-CD3, anti-TCR). Our results show that a previously unrecognized large fraction (at least 30-50%) of all peripheral blood CD4+ T cells can give rise to cytotoxic effector cells. The mode of CD4+ T cell activation (OKT3 hybridoma versus alloantigen) thus determines whether the intrinsic cytotoxic capacity of CD4+ T cells is functionally activated or not.     

CD4+ Leu-8+ T cell supernatant activity that inhibits Ig production.

The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production.     

T cell-induced Ig allotypic suppression in mice. II. Both CD4+ CD8- and CD4- CD8+ T cell subsets from sensitized Igha mice are required to induce suppression of Igh-1b allotype expression

We established the phenotype of T splenocytes (Ts) from Igha/a BALB/c mice sensitized against B splenocytes from the Ighb/b CB20 congenic mice that induce Igh-1b (IgG2a of the Ighb haplotype) suppression. This was achieved by studying the action of anti-T cell subset mAb on the capacity of Ts to induce this chronic allotypic suppression in Igha/b (BALB/c x CB20)F1 mice. The Ts were treated with cytotoxic anti-mouse CD4 or anti-mouse CD8 rat mAb in vitro before their injection into the Igha/b newborns or in vivo after their injection into the Igha/b newborns. Exposure to either anti-CD8 or anti-CD4 mAb in vitro or in vivo leads to loss of the capacity of Ts to induce Igh-1b allotypic suppression. Mixing CD4+-cell-depleted Ts and CD8+-cell-depleted Ts preparations restored the capacity of the cells to induce Igh-1b suppression. Thus, both CD4+ CD8- Ts and CD4- CD8+ Ts are required for the induction of this allotypic suppression. Bone marrow cells and B splenocytes from Igh-1b-suppressed adult Igha/b mice were shown to be able to durably express Igh-1b when transferred into irradiated Igha/a BALB/c hosts whereas whole spleen cells from such donors failed to do it. Abrogation of Igh-1b suppression by in vivo anti-CD8 mAb treatment was achieved in adult Igha/b heterozygotes but with a lower efficiency than in adult Ighb/b homozygotes, all being chronically Igh-1b suppressed. The CD4- CD8+ cell population essential for maintaining this suppression is resistant to in vivo 600 rad irradiation and seems to be slightly inhibited by in vivo administration of free Igh-1b.     

The functional heterogeneity of CD8+ cells defined by anti-CD45RA (2H4) and anti-CD29 (4B4) antibodies.

The monoclonal antibodies, anti-2H4(CD45RA), and anti-4B4(CD29), along with UCHL1-(CD45RO), identify reciprocal populations of CD4 cells with distinct suppressor inducer (CD45RA+CD29-CD45RO-) and helper inducer (CD45RA-CD29+CD45RO+) functions. Although the CD8+ population is known to contain precytotoxic, cytotoxic, suppressor, and some natural killer cells, the exact phenotypic identities of these functional CD8 subsets has not been established. In this study, we tried to determine whether these monoclonal antibodies could distinguish functionally distinct subsets of cells within the CD8+ population. For this purpose, whole T cells or fractionated T cells were sensitized with irradiated allogeneic non-T cells for 6 days, following which, CD8+ or CD8+CD11b- cells were isolated and cellular functions such as suppressor, killer precursor, and killer effector activity were assessed. The results showed that both class I-restricted alloantigen-specific killer effector and killer precursor cells belonged to the CD8+CD11b-CD45RA-CD29+ population. Moreover, these killer effector cells expressed the CTL-associated S6F1 molecule, an epitope of the LFA-1 antigen. In contrast, suppressor effector cells belonged to the CD8+CD11b-CD45RA+CD29- cell population. Although the UCHL1 antigen has been reported to define the CD4+CD29+ helper inducer cell, over 90% of allo-activated CD8+ cells expressed this antigen, whereas only 40-60% of these cells expressed either CD45RA or CD29 antigens. These results suggest that anti-CD45RA and anti-CD29 antibodies may provide useful tools for distinguishing between suppressor effector versus killer effector and killer precursor cells within the CD8+CD11b- population.     

Alloantigen affinity and CD4 help determine severity of graft-versus-host disease mediated by CD8 donor T cells

TCR affinity dictates T cell selection in the thymus and also has a high impact on the fate of peripheral T cells. Graft-vs-host disease (GVHD) is a pathological process initiated by activation of donor T cells after adoptive transfer into an allogeneic recipient. How TCR affinity affects the potential of alloreactive T cells to induce GVHD is unclear. Using alloreactive CD4+ and CD8+ TCR transgenic (Tg) T cells, GVHD models are presented that allow for the visualization of how CD8+ alloreactive T cells behave in response to alloantigens with different TCR affinity in the absence or presence of CD4 help. In a nonmyeloablative transplant model where GVHD lethality is due to marrow aplasia, alloreactive CD8+ TCR Tg T cells induced significantly more severe GVHD in the recipients that express an intermediate-affinity alloantigen than in the recipients that express a high-affinity alloantigen. In a myeloablative transplant model where GVHD lethality is due to epithelium injury, CD8+ TCR Tg cells were also more pathogenic in the recipients with an intermediate-affinity alloantigen than in those with a high-affinity alloantigen. The presence of alloreactive CD4+ TCR Tg cells enhanced the potential of CD8+ TCR Tg cells to cause GVHD in recipients with an intermediate-, but not with a high-, affinity alloantigen. These findings underscore that alloantigen affinity and CD4 help control the fate and pathogenicity of alloreactive CD8+ T cells in vivo.     

Mlsa generated suppressor cells. I. Suppression is mediated by double-negative (CD3+CD5+CD4-CD8-) alpha/beta T cell receptor-bearing cells.

Grafting of cells from B10.D2 (H-2d) donors into H-2 compatible lethally irradiated (DBA/2 x B10.D2)F1 hosts results in a severe graft-vs-host reaction (GVHR), developed against DBA/2 non-H-2 Ag, with only 0 to 10% of animals surviving. This GVHR mortality rate is dramatically reduced (90 to 100% of animals survive) by donor preimmunization against Mlsa determinants. The protection against GVHR correlates with a decreased B10.D2 anti-DBA/2 proliferative response in vitro. Both in vivo and in vitro phenomena are associated with activation of CD5+ suppressor T cells in the spleens of immunized mice. The present work was designed to study the origin of these suppressor cells and to further characterize their phenotype. The results show that significant suppression is not inducible in "B" mice. In contrast, in mice that were only thymectomized or else pretreated in vivo with anti-CD4 or anti-CD8 mAb, the suppressor cells are activated as efficiently as in normal mice. The suppression of GVHR mortality and proliferative responses in vitro is lost after depletion from preimmunized splenocytes of CD5+ T cells and remains unaltered after depletion of CD4+ or CD8+ T cells or both. Depletion of asialo GM1+ cells removes all NK activity, whereas the suppression is decreased only slightly. FACS analysis showed that double-negative (DN) cells from normal and immunized mice contain both CD3+ and CD3- cells; the vast majority of the CD3+ DN T cells express the alpha/beta T cell receptor. Suppression of GVHR and of proliferative responses in vitro are abrogated after elimination of CD3+ cells. These results suggest that Mlsa generated suppressor cells: 1) are derived from post-thymic long-lived T cell precursors; 2) are low asialo GM-1+ but do not exhibit NK activity; 3) belong to a subset of peripheral CD5+ DN T cells bearing a CD3-associated alpha/beta-heterodimer.     

Follicular lymphoma intratumoral CD4+CD25+GITR+ regulatory T cells potently suppress CD3/CD28-costimulated autologous and allogeneic CD8+CD25- and CD4+CD25- T cells

Regulatory T cells (T(R)) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ T(R) that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These T(R) actively suppress both the proliferation of autologous nodal CD8+CD25- and CD4+CD25- T cells, as well as cytokine production (IFN-gamma, TNF-alpha and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these T(R) are also capable of suppressing the proliferation of allogeneic CD8+CD25- and CD4+CD25- T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25- and CD4+CD25- T cells, suggesting that FLN T(R) are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-beta signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-beta in FLN T(R)-mediated suppression.     

Human cytotoxic lymphocytes. IV. Frequency and clonal specificity of CD8+CD16-(Leu2+Leu11-) and CD16+CD3-(Leu11+Leu4-) cytotoxic lymphocyte precursors activated by alloantigen or K562 stimulator cells

Cell sorter-purified CD8+CD16- (Leu2+Leu11-) cytotoxic T cell precursors and CD16+CD3-(Leu11+Leu4-) natural killer (NK) cells were cultured under limiting dilution (LD) conditions with allogeneic stimulator cells or with K562 tumor cells in the presence of exogenous interleukin 2. One out of 100-200 alloantigen-stimulated Leu2+ T cells clonally developed into an alloantigen-specific cytotoxic T cell, but only 1 out of 500-3400 of these cells lysed NK-susceptible K562 target cells. In contrast, 1 out of 2-35 alloantigen-stimulated Leu11+ precursor cells developed into an effector cell that lysed K562, but less than 1 out of 500 of these cells lysed allogeneic Con A blast targets. However, clonal activation of Leu11+ precursor cells under LD conditions did not require alloantigenic stimulator cells. Comparable high frequencies (f = 1/3 to 1/28) of anti-K562 cytotoxic lymphocyte precursors were thus measured when Leu11+ precursor cells were cultured on autologous or K562 feeder cells. As shown by a split culture approach, the vast majority of alloantigen-activated Leu2+ effector cells were highly specific for the stimulating alloantigen (i.e., they did not lyse K562), while the majority of Leu11+ microcultures lysed K562 tumor cells but neither autologous nor allogeneic Con A blast targets. On a quantitative basis, these data show that CD8+CD16- T cells and CD16+CD3-NK cells are two mutually exclusive lymphocyte populations which clonally develop into cytotoxic effector cells specific for alloantigen or K562 target cells, respectively.     

Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells

Studies were performed to determine whether CTL against Toxoplasma gondii-infected cells could be induced in a murine model of T. gondii infection in which CD8+ T lymphocytes have been shown to play a major role in resistance against this parasite. In 51Cr release assays, nylon wool nonadherent spleen cells from BALB/c (H-2d) mice immunized with the temperature-sensitive (ts-4) mutant strain of T. gondii were cytotoxic for T. gondii-infected P815 (H-2d) mastocytoma cells but not for uninfected cells. This cytotoxic activity was remarkably increased after in vitro stimulation with T. gondii-infected syngeneic spleen cells. The effector cells were shown to be CD8+ T lymphocytes, because the cytotoxicity was significantly inhibited by depletion of CD8+ T lymphocytes but not by depletion of CD4+ T lymphocytes. This cytotoxic activity was genetically restricted. Spleen cells from T. gondii-immune BALB/c mice were not cytotoxic for T. gondii-infected EL4 (H-2b) thymoma cells, whereas spleen cells from T. gondii-immune C57B1/6 (H-2b) mice were cytotoxic for T. gondii-infected EL4 cells but not for T. gondii-infected P815 cells. The cytolytic activity of CD8+ T lymphocytes against T. gondii-infected cells might be a mechanism whereby these cells confer resistance against T. gondii.     

Differential expression of CD8 alpha and CD8 beta associated with MHC-restricted and non-MHC-restricted cytolytic effector cells

The differential expression of the alpha and beta chains of the CD8 glycoprotein was examined in three functionally distinct cytolytic effector cell populations: (i) T cells (CD3+ CD56-), (ii) NK cells (CD56+ CD3-), and (iii) non-MHC-restricted T cells (CD56+ CD3+). Twenty-four percent of T cells were CD8+, and they consistently coexpressed both CD8 alpha and CD8 beta. Moreover, CD8+ T cells uniformly expressed high-density CD8 alpha. Forty percent of NK cells were CD8+ but the vast majority (approximately 75%) expressed only CD8 alpha without CD8 beta. In addition, CD8+ NK cells uniformly expressed low-density CD8 alpha. In comparison, 75% of non-MHC-restricted T lymphocytes were CD8+ but they displayed an intermediate phenotype: 60% coexpressed CD8 alpha and CD8 beta while 40% expressed only CD8 alpha. Within this population, CD8 alpha was expressed at high density, similar to that of T cells. Following IL-2 activation, enhancement of non-MHC-restricted cytotoxicity was not associated with any changes in either the quantitative or qualitative pattern of expression of CD8 alpha or CD8 beta by these cells. Addition of either anti-CD8 alpha or anti-CD8 beta mAb did not alter non-MHC-restricted cytotoxicity of either CD56+ CD3- or CD56+ CD3+ effector cells. However, within the CD56+ cell population, non-MHC-restricted cytotoxicity was almost entirely found within the CD8- and CD8 alpha + beta- populations, and both subsets displayed a similar level of killing. In contrast, CD8 alpha+ beta+ cells exhibited very little non-MHC-restricted cytotoxicity. Thus, the coexpression of CD8 alpha and CD8 beta in conjunction with the TCR/CD3 complex appears to characterize MHC restricted cells while the expression of CD8 alpha alone is associated with non-MHC-restricted cytotoxicity. Taken together, these findings suggest that neither CD8 alpha nor CD8 beta is involved in the initial phases of target cell binding or recognition during NK cell-mediated lysis. However, the selective expression of CD8 alpha by a large fraction of non-MHC-restricted effector cells suggests that this antigen may play a different functional role in this unique subset of cytolytic lymphocytes.     

Differential in vitro activation of CD4+CD8- and CD8+CD4- herpes simplex virus-specific human cytotoxic T cells

In order to clarify the differential activation of CD4+ and CD8+ HSV-specific CTL, we compared the characteristics of CTL generated by different methods of in vitro HSV stimulation by treatment of effectors with anti-CD4 and anti-CD8 mAb and C after the elimination of nonspecific cytotoxic effector cells. Cell-free HSV mainly activated CD4+ CTL precursors, whereas HSV-infected fibroblasts were more effective in activating CD8+ CTL precursors than CD4+ CTL precursors. In addition, limiting dilution analyses with enriched T cells from two HSV-seropositive donors revealed that the frequency of HSV-specific CD4+ CTL precursors responsive to stimulation with free HSV was approximately 1/4,000 to 6,000 CD4+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/19,000 to 22,000 CD4+ T cells. Conversely, the frequency of CD8+ CTL precursors in peripheral blood responsive to stimulation with free HSV was approximately 1/28,000 to 30,000 CD8+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/10,000 to 11,000 CD8+ T cells. The present data suggest that generalized viral infection due to cell-free viruses is fought mainly by CD4+ CTL, which have previously been reported to possess both cytotoxicity and helper function, and that localized viral infection on HLA class II-negative fibroblasts is prevented from spreading to adjacent cells mainly by CD8+ CTL. Such differential activation of CD4+ and CD8+ CTL seems probable when considering the protective mechanisms against viral infection.     

Heterogeneous mechanisms of human cytotoxic T lymphocyte generation. I. Differential helper cell requirement for the generation of cytotoxic effector cells from CD8+ precursor subpopulations.

The subpopulations of CD8+ T cells defined by CD45RA Ag expression have been hypothesized to represent cells varying in their relative maturation along a common, activation-dependent differentiation pathway. Previous studies have shown that both the CD8+CD45RA+ and CD8+CD45RA- subsets contain precursor cells capable of developing into alloreactive CTL. In the current study, we have examined the mechanisms involved in the generation of CTL effector cells from these two CD8+ subsets. Purified CD8+CD45RA+ or CD8+CD45RA- cells were stimulated with allogeneic non-T cells, either alone or in the presence of CD4+ Th cells. Although the generation of CTL from CD8+CD45RA- precursor cells consistently required the presence of CD4+ Th cells, cytotoxic effector cells could be generated from CD8+CD45RA+ precursor cells in the absence of CD4+ cells. Several lines of evidence indicated that the helper cell-independent generation of cytotoxic effector cells from CD8+CD45RA+ precursors resulted from the unique ability of this subset to produce and use IL-2 in an autocrine fashion: 1) exogenous IL-2 could replace the effects of CD4+ helper cells for either CD8+ subset; 2) the helper cell-independent functional maturation of CD8+CD45RA+ cells could be blocked by anti-CD25 or anti-IL-2 antibodies; and 3) CD8+CD45RA+ cells produced IL-2 after activation with allogeneic cells. The finding that precursors for helper cell-independent CTL generation are restricted to the CD8+CD45RA+ subset suggests that this capability may vary as a function of the maturation of CD8+ cells.     

Alloantigen-specific T suppressor-inducer and T suppressor-effector cells can be activated despite blocking the IL-2 receptor

To determine IL-2 requirement for activation of suppressor cells, PBMC were primed in one-way MLR in the presence of 10 micrograms/ml anti-IL-2R beta-chain antibody 2A3 (CD25) or control antibody, then irradiated and added as regulators in a fresh MLR. Cells primed in the presence of antibody 2A3 suppressed the proliferative response to fresh autologous lymphocytes to specific alloantigen but had no effect on the response to cells from third party donors. Priming in the presence of an antibody of irrelevant specificity induced only limited suppressor activity. Activated suppressor cells did not show cytolytic activity specific for the stimulators when tested at the time of the suppressor cell assay. To identify the subset(s) responsible for suppression, cells primed in the presence of antibody 2A3 were separated into CD4+/CD45RA+, CD4+/CD45RA-, and CD8+ subsets, which were irradiated and then tested. The suppressive activity was found predominantly in the CD4+/CD45RA+ subset, whereas CD8+ cells had some activity and CD4+/CD45RA- cells had none. No subset suppressed the response of autologous cells to third-party cells. When primed CD4+/CD45RA+ cells were cocultured with fresh autologous lymphocytes depleted of CD8+ cells, no suppression was observed, indicating that, although the CD4+/CD45RA+ cells can function as inducers of suppressors, they cannot function as suppressor-effectors. Conversely, CD8+ cells activated in MLR in the presence of 2A3 caused suppression, regardless of whether the fresh autologous responder population contained CD8+ cells. CD4+/CD45RA+ and CD8+ subsets isolated after priming in the presence of 2A3 also demonstrated Ag-specific suppression in the generation of cytotoxic T lymphocytes whereas CD4+/CD45RA- cells had no activity. Our data are consistent with the model that suppression of alloreactivity requires the cooperation of two types of cells, a CD4+/CD45RA+ suppressor-inducer and a CD8+ suppressor-effector population. Activated Tsi and fresh Tse or activated Tse alone can suppress lymphocyte proliferation and generation of CTL in response to specific Ag. Activation of Ag-specific T suppressor-inducer and T suppressor-effector cells appears to be relatively IL-2 independent and presumably require one or more other growth factors.     

Intrahepatic, nucleocapsid antigen-specific T cells in chronic active hepatitis B

Hepatitis B core antigen (HBcAg)-specific T cell lines were established from hepatic lymphomononuclear cells derived from five patients with chronic active hepatitis B. No hepatitis B virus envelope antigen-specific cell lines were established. Proliferation in response to recombinant and native HBcAg, but not to native hepatitis B surface antigen containing the pre-S(2) region, confirmed the specificity of the five T cell lines. All cell lines represented mixed populations of CD4+ and CD8+ T cells. The CD4+ subset provided antigen-specific help to autologous B cells with respect to anti-HBc production and to CD8+ cells with regard to HBcAg-induced proliferation and suppressor activity. The CD8+ subset contained suppressor cells that selectively inhibited the proliferative response of autologous HBcAg-specific CD4+ cells without inhibiting CD4+ cells of unrelated specificity (tetanus toxoid). Moreover, the CD8+ cells were also capable of suppressing HBcAg-stimulated antibody to HBcAg production without showing inhibition of total immunoglobulin production stimulated by pokeweed mitogen. The cytotoxic potential of the T cell lines was established in a lectin-dependent cytotoxicity system; natural killer cytotoxicity was completely absent. Our data suggest that the lesional T cells present at the site of hepatocellular injury in chronic active hepatitis B are primarily HBcAg-specific lymphocytes of the helper and suppressor/cytotoxic phenotypes and that both are functionally competent.