Antigenicity and immunogenicity of allogeneic retinal transplants.

The transplantation of neuronal cells and tissues represents a promising approach for the treatment of incurable neurodegenerative diseases. Indeed, it has been reported recently that retinal transplantation can rescue photoreceptor cells and delay age-related changes in various retinal layers in rodents. However, retinal grafts deteriorate progressively after placement in recipients' eyes. Here we investigated whether a host's immune response elicited toward the graft contributes to its deterioration. Using an ELISA spot assay, we measured T cell responses to retinal tissues placed in the vitreous cavity of syngeneic and allogeneic mice. We found that allogeneic retinas induced potent alloimmune responses mediated by T cells secreting type 1 cytokines (IFN-gamma and IL-2). No response was found in mice engrafted with syngeneic retinas. In addition, all syngeneic retinal grafts displayed no signs of tissue damage (at 55 days), while the majority of allogeneic retinas deteriorated as early as 12 days after placement. Next, we showed that anti-donor responses occurred within two phenotypically and functionally distinct T cell subsets: CD4+ T cells secreting IL-2 and CD8+ T cells producing IFN-gamma. Importantly, CD4+ T cells were necessary and sufficient to cause graft deterioration, while CD8+ T cells did not contribute to this process.

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.

Immunosuppressive factor from liver induces apoptosis in thymoma EL-4 cells but not normal MHC class II-specific T lymphocytes.

An endogenously produced immunosuppressive factor (ISFnp, immunosuppressive factor-neutral protein), inducing a decrease in viability of thymoma EL-4 cells in vitro, was isolated from murine liver using ion exchange, gel filtration and hydrogen-bonding chromatography. Polyclonal rabbit antibodies against this factor were developed and attached to periodate-activated Sepharose CL-6B. The immunoaffine sorbent obtained significantly depleted the biological activity of ISFnp from tested fractions. The factor shows liver-specific location, an M(r) of about 70-80 kDa and consists of 2 subunits (40 and 42 kDa) as determined by SDS-PAGE and Western blotting. ISFnp induced DNA degradation in EL-4 cells similar to the cleavage of DNA onto olygonucleosomal fragments in dexamethasone-treated thymocytes. This DNA degradation preceded lysis of thymoma cells, suggesting an induction of apoptosis in ISFnp-treated EL-4 cells. Addition of the factor into primary mixed lymphocyte culture (MLC) strongly inhibited proliferative response but failed to induce any decrease in the ability of normal MHC class II-specific alloreactive cells to respond in the secondary MLC. Moreover, addition of ISFnp into primary MLC on the peak of proliferative response resulted in augmentation of secondary responses of primed cells as compared with the same quantities of primed cells from untreated cultures. These results suggest a possible role of liver both in deletion of transformed clones of T lymphocytes and formation of allospecific memory T cells.

Induction of T-cell response to cryptic MHC determinants during allograft rejection.

The presentation of MHC peptides by recipient and donor antigen presenting cells is an essential element in allorecognition and allograft rejection. MHC proteins contains two sets of determinants: the dominant determinants that are efficiently processed and presented to T cells, and the cryptic determinants that are not presented sufficiently enough to induce T-cell responses in vivo. In transplanted mice, initial T-cell response to MHC peptides is consistently limited to a single or a few immunodominant determinants on donor MHC molecule. However, in this article we show that under appropriate circumstances the hierarchy of determinants on MHC molecules can be disrupted. First, we observed that gamma IFN can trigger de novo presentation of cryptic self-MHC peptides by spleen cells. Moreover, we showed that allotransplantation is associated with induction of T-cell responses to formerly cryptic determinants on both syngeneic and allogeneic MHC molecules. Our results suggest that cross-reactivity and inflammation are responsible for the initiation of these auto- and alloimmune responses after transplantation.

[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.

Cholera toxin, E. coli heat-labile toxin, and non-toxic derivatives induce dendritic cell migration into the follicle-associated epithelium of Peyer's patches.

The follicle-associated epithelium (FAE) of Peyer's patches (PPs) transports antigens and microorganisms into mucosal lymphoid tissues where they are captured by subepithelial dendritic cells (DCs). Feeding of cholera toxin (CT) induced migration of subepithelial DCs to interfollicular T-cell areas within 24 h. This study investigated short-term effects of CT, Escherichia coli heat-labile toxin, and non-toxic derivatives on DC migration. CT or CTB injected into ligated intestinal loops induced significant increase in CD11c+ DCs within the FAE within 90 min. In mice fed CT intragastrically, DC numbers in the FAE increased by 1 h, were maximal by 2 h, declined between 8 and 12 h, and were reversed by 24 h. Feeding of native LT, recombinant CTB, dibutyryl cyclic AMP, and to a lesser extent mutated CT(E29H) or mutated LT(R192G) had the same effect. Thus, both A and B subunits of enterotoxins, presumably acting through distinct signaling pathways, may promote capture of incoming antigens and pathogens by PP DCs.

[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.

[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.

CD4+ T cell responses to self- and mutated p53 determinants during tumorigenesis in mice.

We analyzed CD4+ T helper responses to wild-type (wt) and mutated (mut) p53 protein in normal and tumor-bearing mice. In normal mice, we observed that although some self-p53 determinants induced negative selection of p53-reactive CD4+ T cells, other p53 determinants (cryptic) were immunogenic. Next, BALB/c mice were inoculated with J774 syngeneic tumor cell line expressing mut p53. BALB/c tumor-bearing mice mounted potent CD4+ T cell responses to two formerly cryptic peptides on self-p53. This response was characterized by massive production of IL-5, a Th2-type lymphokine. Interestingly, we found that T cell response was induced by different p53 peptides depending upon the stage of cancer. Mut p53 gene was shown to contain a single mutation resulting in the substitution of a tyrosine by a histidine at position 231 of the protein. Two peptides corresponding to wt and mutated sequences of this region were synthesized. Both peptides bound to the MHC class II-presenting molecule (Ed) with similar affinities. However, only mut p53.225-239 induced T cell responses in normal BALB/c mice, a result strongly suggesting that high-affinity wt p53.225-239 autoreactive T cells had been eliminated in these mice. Surprisingly, CD4+ T cell responses to both mut and wt p53.225-239 peptides were recorded in J774 tumor-bearing mice, a phenomenon attributed to the recruitment of low-avidity p53.225-239 self-reactive T cells.

CD28-independent costimulation of T cells in alloimmune responses.

T cell costimulation by B7 molecules plays an important role in the regulation of alloimmune responses. Although both B7-1 and B7-2 bind CD28 and CTLA-4 on T cells, the role of B7-1 and B7-2 signaling through CTLA-4 in regulating alloimmune responses is incompletely understood. To address this question, we transplanted CD28-deficient mice with fully allogeneic vascularized cardiac allografts and studied the effect of selective blockade of B7-1 or B7-2. These mice reject their grafts by a mechanism that involves both CD4(+) and CD8(+) T cells. Blockade of CTLA-4 or B7-1 significantly accelerated graft rejection. In contrast, B7-2 blockade significantly prolonged allograft survival and, unexpectedly, reversed the acceleration of graft rejection caused by CTLA-4 blockade. Furthermore, B7-2 blockade prolonged graft survival in recipients that were both CD28 and CTLA-4 deficient. Our data indicate that B7-1 is the dominant ligand for CTLA-4-mediated down-regulation of alloimmune responses in vivo and suggest that B7-2 has an additional receptor other than CD28 and CTLA-4 to provide a positive costimulatory signal for T cells.