Differential effect of CD8(+) and CD8(-) dendritic cells in the stimulation of secondary CD4(+) T cells.

Dendritic cells (DC), in their role in initiation of the adaptive immune response, have been extensively studied for their capacity to interact and stimulate naive T cells. Subsets of mature murine DC isolated directly from the spleen have been shown to differ in their ability to induce proliferative responses in both primary CD4(+) and primary CD8(+) T cells; the myeloid-related CD8alpha(-) DC induce a more intense or prolonged proliferation of naive T cells than do the lymphoid-related DC bearing CD8alpha despite similar expression of MHC and co-stimulatory molecules. Here we examine the interaction of these DC subpopulations with T cells already in the activated or memory state which are known to have greater sensitivity to antigen stimulation and bear receptors with increased capacity for signal transduction. We show that influenza virus-specific CD4(+) T cell clones and splenic T cells from peptide-primed animals proliferated in response to antigen presented by separated splenic CD8(-) DC. In contrast, these T cells showed only weak, if any, proliferation in response to CD8(+) DC despite observable cluster formation in the cultures. The differential between the two DC types in inducing proliferation was even more pronounced than previously seen with primary T cells and did not reflect differential longevity of the DC in culture, altered response kinetics or deviation from IL-2 to IL-4 induction with CD8(+) DC, but was related to the levels of IL-2 induced. The deficiency in the CD8(+) DC was not overcome by using infectious virus rather than synthetic peptide as the antigen source. These results show that lymphoid-related CD8(+) splenic DC, despite their mature phenotype, fail to provide appropriate signals to secondary CD4(+) T cells to sustain their proliferation.

Regulation of T cell cytokine production by dendritic cells.

Previous work has established that the dendritic cells (DC) of mouse spleen regulate the IL-2 production, and hence the extent of proliferation, of the CD8 T cells they activate. It is now reported here that interaction of primary CD8 T cells with splenic CD8alpha- DC induced much higher production of IL-3, IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as IL-2, than did interaction with CD8alpha+ splenic DC. Furthermore, the CD8alpha- DC also induced higher levels of IL-2, IL-3 and IL-10 production in primary CD4 T cells, compared with that induced by CD8alpha+ DC. These quantitative differences did not involve qualitative shifts in the type of cytokine produced. Interleukin-4 production remained low in all the primary T cell cultures and restimulation experiments in secondary cultures did not reveal any bias in the cytokine production profile. When exogenous IL-2 was added to the primary cultures to ensure equal proliferation in response to CD8alpha- or CD8alpha+ DC, the higher level of production of IL-3, IFN-gamma and GM-CSF induced by CD8alpha- DC was maintained. Thus, this general control of T cell cytokine production by splenic DC involves factors additional to those that govern activation of T cells into cell cycle.

The geometry of synthetic peptide-based immunogens affects the efficiency of T cell stimulation by professional antigen-presenting cells.

In the pathway leading to antibody production there are two points at which CD4(+) T(h) cells need to be recruited. The first of these is priming of T cells by their interaction with dendritic cells (DC) bearing antigen presented on MHC class II molecules and the second is the collaborative interaction of these primed T cells with B cells presenting the same antigen. We have previously shown that the configuration of T and B cell determinants within synthetic peptide immunogens can greatly influence the amount of immunogen required to produce an antibody response. Here we investigate whether the difference in potency of different immunogens is related to their ability to be presented by either DC or B cells. We show that determinants in a branched configuration, which are the most efficient at eliciting antibody in vivo, are presented to T cell clones by splenic CD8(-) DC 10-fold more efficiently than the corresponding determinants in a tandem linear arrangement. B cells also showed preferential presentation of branched immunogens to one T cell clone but in contrast to DC, not to a second T cell clone, indicating differences between the two antigen-presenting cell types. We also show that branched immunogens have a greater stability in serum compared to linear peptides, which may further enhance the differences in their in vivo potency.

DEC-205 as a marker of dendritic cells with regulatory effects on CD8 T cell responses.

We have previously reported that a population of lymphoid-related CD8alpha(+) DEC-205(+) dendritic cells (DC) from mouse spleen have 'regulatory' effects on the T cells they activate. CD8 T cells produce IL-2 and give a sustained proliferative response to allogeneic CD8alpha(-) DEC-205(-) splenic DC, but produce little IL-2 and give a limited response to allogeneic CD8(+) DEC-205(+) splenic DC. Although CD8alpha and DEC-205 correlate closely among splenic DC, lymph nodes (LN) include a large population of CD8alpha(low) DEC-205(high) DC. By i.v. transfer of purified thymic early lymphoid precursors into irradiated recipient mice we now demonstrate that these CD8alpha(low) but DEC-205(high) LN DC can be the progeny of a lymphoid precursor population, apparently corresponding to the CD8alpha(high) DEC-205(high) DC progeny of the same precursors in spleen and thymus. By culture of the separated, purified DC with allogeneic CD8 T cells we demonstrate that the CD8alpha(low) DEC-205(high) DC of LN are also functionally equivalent to the CD8alpha(high) DEC-205(high) DC of spleen. Therefore, DEC-205 but not CD8alpha serves to segregate functionally distinct DC types in LN. However, DC isolated from the spleens of genetically manipulated DEC-205(null) mice and separated on the basis of CD8alpha expression have a similar capacity to stimulate CD8 T cells as their heterozygous littermate controls, with the CD8alpha(+) but now DEC-205(null) DC still giving restricted responses. In conclusion, high expression of DEC-205 appears to be a good marker of the lymphoid-related regulatory type of DC, but DEC-205 itself is not responsible for transmitting negative signals to the T cells.

Does the IL-2 receptor alpha chain induced on dendritic cells have a biological function?

The IL-2 receptor (IL-2R) alpha chain (CD25), but not the IL-2R beta chain, is induced on dendritic cells (DC) by brief periods of culture. To test if this IL-2R alpha is important for DC function, DC were isolated from the spleens of mutant mice with the IL-2R alpha gene disrupted and compared with normal DC for ability to stimulate proliferation of allogeneic CD4 and CD8 T cells in culture. The IL-2R alpha null DC and the normal DC produced nearly identical proliferative responses from CD4 and from CD8 T cells. When the CD8 alpha+ and CD8 alpha- subsets of the IL-2R alpha null DC were separated, they also produced proliferative responses similar to that of their normal DC counterparts. Overall there was no evidence that the inducible IL-2R alpha on DC was required for DC development, for stimulation of T cells or for regulation of T cell responses.

Are CD8+ dendritic cells (DC) veto cells? The role of CD8 on DC in DC development and in the regulation of CD4 and CD8 T cell responses.

The CD8-expressing dendritic cells (DC) present in mouse spleen have been shown to have a regulatory effect on the CD4 and CD8 T cells they activate, restricting subsequent T cell proliferation by either inducing apoptotic T cell death (CD4 T cells) or by limiting endogenous cytokine production (CD8 T cells). To determine the role of the CD8 molecule itself in these regulatory phenomena, the DC from CD8 null mice were studied. The DC marker DEC-205 (NLDC 145) was used as a surrogate marker for CD8, since the expression of these two molecules on splenic DC was closely correlated. DC levels were normal, and the incidence of DEC-205+ and DEC-205- DC was normal in CD8 null mice, indicating that the absence of CD8 did not affect DC development. The proliferative response of T cells to allogeneic DEC-205+ DC from either CD8-/- or CD8+/+ mice was similar and was much less than the response to DEC-205- DC from these mice. This applied to both the CD4 and the CD8 T cell responses. Thus the lack of the CD8 molecule did not affect the stimulatory or regulatory properties of the DC. The regulatory CD8+ DEC-205+ DC therefore differ in that respect from antigen-presenting 'veto' cells, where CD8 itself is involved in transmitting negative signals to the T cells. DEC-205 may prove to be a more pertinent marker of the regulatory DC population.

Dendritic cells and T lymphocytes: developmental and functional interactions.

Dendritic cells (DCs) are specialized for presentation of antigen to T cells and are essential for primary T cell activation. Although DCs are generally considered to be myeloid derived, we now have evidence that a subgroup are of lymphoid origin. In particular, the DCs of the adult mouse thymus appear to be derived from the same early, lymphoid-restricted precursor cells that generate T lymphocytes. Purified early thymic T precursors have the capacity to produce T cells, B cells, NK cells and DCs, but not myeloid cells, on transfer to irradiated recipients. They also produce thymic DCs on culture with a mix of cytokines; this mix does not include GM-CSF, needed to generate myeloid-derived DCs. A subgroup of DCs in other lymphoid organs, which like thymic DCs express CD8 as an alpha alpha homodimer, may likewise be of lymphoid origin. These CD8+ DCs in mouse spleen differ functionally from the conventional CD8+ DCs. CD8+ DCs efficiently activate CD4+ T cells but then kill them via Fas ligand on the DC surface. CD8+ DCs efficiently recruit CD8+ T cells into the cell cycle, but their proliferation is then restricted by an inadequate production of interleukin 2. This subgroup of CD8+ DCs therefore appears to have a regulatory role.

The nature of the signals regulating CD8 T cell proliferative responses to CD8alpha+ or CD8alpha- dendritic cells.

The CD8alpha(-)-expressing dendritic cells (DC) of mouse spleen have been shown to be poor inducers of interleukin (IL)-2 production by CD8 T cells when compared to the CD8- DC. As a consequence, CD8 T cells give a more prolonged proliferative response to CD8- DC than to CD8+ DC. The possible mechanisms underlying these functional differences in DC subtype have been investigated. Inadequate co-stimulation did not underlie the poor T cell response to allogeneic CD8+ DC. Equivalent levels of B7-1 (CD80) and B7-2 (CD86) were found on the two DC subtypes and co-stimulator assays did not reveal any functional differences between them. Although CD8+ DC were found to die more rapidly in culture than CD8- DC, this did not explain their reduced stimulatory ability. Neither prolonging DC survival in culture nor renewing the stimulator cells by repeated addition of freshly isolated DC had any significant effect on the T cell responses. Furthermore, later addition to the cultures of DC of the opposite type to the initiating DC did not reverse or eliminate the differential response to the initiating DC. The role of DC-derived soluble factors was examined by addition to the cultures of supernatants derived from freshly isolated or stimulated DC of the opposite type. This neither enhanced the poor stimulatory capacity of CD8+ DC nor inhibited the stimulation by CD8- DC. Furthermore, addition of a series of cytokines that might have been produced by the DC did not eliminate the differences in T cell proliferation. Only the addition to the cultures of the growth factors IL-2 and IL-4 overcame the stimulatory difference between the two DC populations, confirming that the difference in T cell proliferative responses was a consequence of differences in induced cytokine production. The difference in the response of CD8 T cells to CD8+ and CD8- DC is therefore determined by direct DC-T cell contact during the earliest stages of the culture and involves an undetermined and possibly new signaling system.

A subclass of dendritic cells regulates the response of naive CD8 T cells by limiting their IL-2 production.

Previous work indicated that a subclass of mouse spleen dendritic cells (DC), those bearing CD8alpha, expresses the Fas ligand and restricts peripheral CD4 T cell responses by initiating Fas-mediated apoptosis. To determine whether a similar regulation applies to CD8 T cells, they were purified from normal or from TCR-transgenic mice, and then cultured with purified splenic CD8+ DC or CD8- DC presenting either alloantigens or the specific Ag for the TCR transgene. In all systems studied, the proliferative response of CD8 T cells was markedly less on stimulation with CD8+ DC compared with conventional CD8- DC. However, the basis of this restricted proliferation in response to CD8+ DC was totally different for CD8 T cells than for CD4 T cells. The reduced proliferation of CD8 T cells occurred later in the response than with CD4 T cells. In contrast with CD4 T cells, the reduced proliferation of CD8 T cells occurred even with T cells from Fas-deficient Ipr mice, or with DC from Fas ligand-deficient gld mice, indicating that Fas-induced apoptosis was not involved. Also, in contrast with CD4 T cells, the reduced proliferation of CD8 T cells was completely reversed by the addition of exogenous IL-2. Furthermore, cultures of CD8 T cells with CD8+ DC were found to be deficient in IL-2 production. Accordingly, although CD8+ DC are very efficient at stimulating CD8 T cells into cell division, they are deficient at stimulating endogenous cytokine production. The implications of these different DC regulatory systems are discussed.

[The induction of alloantigen-specific cytotoxic T-lymphocytes by the intravenous immunization of mice]. / Induktsiia alloantigen-spetsificheskikh tsitotoksicheskikh T-limfotsitov pri vnutrivennoi immunizatsii myshei.

It is shown possible to induce specific cytotoxic T-lymphocytes (CTL) in the course of intravenous (i.v.) immunization of Balb/c mice by 2000-rad-irradiated allogeneic C57Bl/6 splenocytes in a dose of 9 x 10(7). The induced CTL express Thy1.2+L3T4-Ly2+ cell surface markers. No correlation was observed between the level of cytotoxic activity and the ability to inhibit proliferation in the population of lymphocytes primed by i.v. immunization.

Induction of alloantigen-specific cytotoxic and suppressor activities of lymphocytes by intravenous immunization of mice when donor and recipient differ in class II MHC molecule.

Induction of alloantigen-specific cytotoxic and suppressor activities of lymphocytes was studied upon intravenous immunization of recipients C57Bl/6 with B6.C-H-2bm12 donor's splenocytes irradiated with 2000 rads and introduced in a dose of 9 x 10(7). A possibility was shown to induce a specific cytotoxic and suppressor activities may be induced individually. We studied a possibility to detect the suppressor activity of immune splenocytes in the reaction of skin graft rejection. No significant prolongation of the B6.C-H-2bm12 skin graft life was noted in C57Bl/6 recipients, irradiated with 2000 rads, under the action of adoptive transfer of the immune syngenic splenocytes in comparison with the grafts life in recipients received the adoptive transfer of intact splenocytes.

[The stimulation of the proliferative reaction of immune splenocytes by monoclonal antibodies to the beta-chain of the LFA-1 molecule can lead to an increase in their cytotoxicity]. / Stimuliatsiia proliferativnoi reaktsii immunnykh splenotsitov monoklonal'nymi antitelami k beta-tsepi molekuly LFA-1 mozhet privodit' k usileniiu ikh tsitotoksichnosti.

A system for investigation of relationship between proliferation and cytotoxicity of immune splenocytes fractions stimulated by monoclonal antibodies (mAb) to beta-chain LFA-1 molecule was elaborated. To obtain different proportional contents of effector cells in populations, the immune splenocytes were eluted from donor, third-party, and recipient macrophage monolayers. Cytotoxic indices of effector splenocyte fractions were compared before and after their proliferation in mixed lymphocyte culture. The results do not exclude the role of proliferation in increasing the effector cells proportions in populations stimulated by mAb to beta-chain LFA-1 molecule.

[Monoclonal antibodies to the beta-chain of the LFA-1 molecule promote an increase in the cytotoxic index of effector lymphocytes and stimulate their proliferation]. / Monoklonal'nye antitela k beta-tsepi molekuly LFA-1 sposobstvuiut povysheniiu tsitotoksicheskogo indeksa éffektornykh limfotsitov i stimuliruiut ikh proliferatsiiu.

The influence of monoclonal antibodies (mAb) to LFA-1 molecule beta-chain on the functional activities of immune splenocyte fractions which were eluted from donor, third party and recipient macrophage monolayers was studied. The splenocyte fractions were treated with the mAb and tested for cytotoxicity and proliferation. MAb to LFA-1 molecule beta-chain (without co-stimulation by other antibodies) were found to stimulate the immune response of the splenocyte fractions in both functional tests. The observed effect might be non-specific. The relationship between the stimulation of proliferative and cytotoxic responses of the immune splenocyte fractions and the treatment with mAb of LFA-I molecule beta-chain remains obscure.

[Effect of the Ly2 molecule on the function of alloantigen-specific effector cytotoxic T-lymphocytes as a function of the variability of their receptors' affinity]. / Vliianie molekuly Ly2 na funktsiiu alloantigenspetsifichnykh éffektornykh tsitotoksicheskikh T-limfotsitov v zavisimosti ot variabel'nosti srodstva ikh retseptorov.

To study T cell receptors' affinity alloantigen-specific anti-K cytotoxic T-lymphocytes (CTL) were divided on fractions by elution from donor K and third-party macrophage monolayers. The functional activity of CTL was suppressed by anti--Ly2 monoclonal antibodies (mAb), 51-Cr-labelled transformed fibroblasts (L-cells) transferred with the H-2K gene were used as targets. The results demonstrate that primary CTL enriched on third-party macrophage monolayers are the most sensitive to anti-Ly2 mAb. T-cells exhausted on third-party monolayer and then enriched on donor monolayer were resistant to treatment with the mAb. Secondary CTL enriched on donor monolayer were resistant to treatment with anti-Ly2 mAb even should they were not exhausted on third-party monolayer. These results show that Ly2 (CD8) molecule plays an essential role in the interaction of CTL with MHC class I molecule only if T-cell receptor has low affinity.

Intravenous injection of allogeneic cells can prime T-lymphocytes with cytotoxic activity.

The possibility of specific cytotoxic T-lymphocyte (CTL)2 induction was shown upon intravenous (i.v.) immunization of mice with 9 x 10(7) irradiated (2000 rad) allogeneic splenocytes. The induced CTL express the cell surface markers Thy1.2+, L3T4- and Lyt2+. No correlation between the level of cytotoxic activity and the ability to inhibit proliferation was shown in populations of lymphocytes, primed both by i.v. immunization and in mixed lymphocyte culture (MLC). The possible role of cytotoxic activity in down-regulation of the immune response is discussed.

[Thymectomy and splenectomy of adult mice have no influence on the level of suppressor activity of T-lymphocytes specific to antigens of H-2 complex]. / Timéktomiia i splenéktomiia vzroslykh myshei ne vliiaiut na uroven' supressornoi aktivnosti T-limfotsitov, spetsifichnykh k antigenam H-2 kompleksa.

I.v. immunization of mice with irradiated (1500 rad) allogeneic spleen cells induces T cells specific to histocompatibility antigens and capable to suppress proliferation in mixed lymphocyte cultures. The lymph node cells appeared to be almost as capable as spleen cells of producing suppression. Splenectomy experiments showed that the spleen is not essential for induction of suppressive activity of T cells. The precursors of T cells with suppressive activity do not belong to the pool of short-living cells as they are insensitive to adult thymectomy, produced 6 weeks before immunization.

Special differentiation requirements of original and enriched secondary cytotoxic T lymphocyte precursors (pCTL-2 memory cell) specific for the class I histocompatibility molecule.

The in vivo-induced pCTL-2 cells of the L3T4- Lyt-2+ phenotype specific for the H-2Kb molecule are converted into effector CTLs in mixed lymphocyte culture (MLC) in the presence of heat-treated donor stimulators much more efficiently when the donor and recipient differ from each other, not only in the major histocompatibility complex (MHC) class I (H-2Kb) (anti-B10.MBR B10.AKM) but also in the MHC classes I + II, i.e., Kb + Ib (anti-C57BL/6 B10.D2 (R101)). The differentiation of original pCTL-2 is enhanced as the result of a synergistic action of the Kb alloantigen and rIL-2 in small doses. The anti-Kb pCTL-2, after their separation from helper T cells non-adherent to the macrophage donor monolayer and their subsequent elution from it, give rise to pronounced differentiation in simplified conditions over 3 days in monoculture in the absence of both stimulators and rIL-2. It is suggested that spontaneous and highly efficient specific differentiation of the eluted pCTL-2 is due to a dramatically enhanced secretion of the CTL differentiation factor by pCTL-2 themselves, and in addition, rIL-2 can also contribute to the secretion of this factor.

Difference between antigen-binding receptor repertoires in effector cytotoxic T lymphocytes and their secondary precursors (memory cells) specific to H-2Kb.

The fine specificity of antigen-binding receptors was compared in pCTL-2 and secondary effector CTL (cytotoxic T lymphocytes) induced in vivo with the H-2Kb alloantigen in recombinant inbred mice. The lymphocyte preparations were enriched by elution from macrophage monolayers of various origins, including the donor (B6 strain), the H-2Kb mutant bm1, the H-2Kk allele B10.A(4R) and the recipient strain B10.D2(R101) as a control. Anti-Kb pCTL-2 eluted from third-party bm1 or B10.A(4R) monolayers gave rise to CTL progeny that lysed, equally well, both donor TC and those third-party TC from whose monolayer the pCTL-2 had been eluted, but which were unable to lyse irrelevant third-party TC. The lytic activities of secondary CTL whose precursors had been eluted from bm1 or B10.A(4R) monolayers were 6 and 12 times lower, respectively, than pCTL-2 eluted from the donor monolayer. Opposite results were shown for receptors of enriched secondary anti-Kb effector CTL. Irrespective of their elution source, whether donor, mutant or allele variant, the eluted effector CTL were able to lyse the donor TC to a similar degree and much more than the given third-party TC; moreover, they retained cross-reactivity in all cases. It is suggested that CTL receptors are homogeneous in specificity for a whole composite immunodominant epitope and differ from each other only in the affinity/lability of the combining site. In contrast, pCTL-2 can be separated into fractions: receptors of each fraction are strictly specific (with high affinity) to a particular portion of the same composite CTL epitope. It seems likely that the pCTL-2 receptor antigen-binding site is modified during pCTL-2 in vivo differentiation into effector CTL.