Expression of T-cell differentiation antigens on activated Thymocytes

The expression of Thy 1, TL, Lyt1, and Lyt2 antigens on resting and proliferating thymocytes activated by concanavalin A in the presence of interleukin 2 has been studied by conventional complement-dependent cytotoxicity assay. The predominant population of resting thymocytes has a TL⁺Lytl⁺Lyt2⁺ phenotype while the predominant population of proliferating thymocytes has a TL — Lytl⁺Lyt2⁺ phenotype. Using several separation procedures such as agglutination by peanut lectin, BSA density gradient centrifugation, and pretreatment with high dilutions of anti-H-2 serum it was impossible to obtain a 100% pure population of TL⁺Lytl⁺Lyt2⁺ cells, suggesting that the population of resting immature thymocytes contains small subpopulations of phenotypically differentiated cells. The population of proliferating thymocytes is also phenotypically heterogenous and contains cells bearing all phenotypes that were described for different stages of T-cell differentiation, including TL⁺Lytl⁺Lyt2^? and TL⁺Lytl^?Lyt2⁺ with the following approximate frequency: TL⁺Lytl⁺Lyt2⁺—27%, TL⁺Lytl⁺Lyt2^?—8%, TL⁺Lyt1^?Lyt2⁺—4%, TL^?Lytl⁺Lyt2⁺—45%, TL^?Lyt1⁺Lyt2^?—13%, TL^?Lyt1^?Lyt2⁺—3%.     

Different functions of subsets of effector T cells in murine influenza virus infection

Enriched preparations of secondary effector T cells to influenza virus were tested for their in vivo biological function by adoptive transfer to mice 24 hr after an intranasal inoculation of infectious influenza virus. One class of cells which were Lyt 1⁺2^?3^?, I region-restricted, and could mediate DTH reaction failed to reduce lung virus titers 5 days after transfer and caused a higher mortality rate in the recipient mice than in the controls. A second class of cells which were Lyt 1^?2⁺3⁺, K,D region-restricted, and were cytotoxic and could mediate DTH activity substantially reduced lung virus titers 5 days after transfer. The influx of mononuclear cells to the lungs after adoptive cell transfer was measured by injection of [¹²⁵I]UdR 24 hr prior to harvest of lung cells, using both infected CBA and athymic BALB/ c nude (nu/nu) mice as recipients. I region-restricted cells caused increased cellular infiltration which was very marked in athymic mice. It was concluded that this reaction significantly contributed to the observed immunopathology in infected mice. Transfer of K,D region-restricted cells reduced the cellular infiltration in infected CBA mice and caused only a slight increase in infected athymic mice. The evidence supported the concept that the second class of cells exerted their protective (antiviral) effect in vivo by direct lysis of virus-infected cells rather than by liberation of lymphokines.     

Adoptive transfer of delayed-type hypersensitivity to Sendai virus. I. Induction of two different subsets of T lymphocytes which differ in H-2 restriction as well as in the Lyt phenotype

This paper investigates kinetics and antigenic and genetic requirements for transfer of delayed-type hypersensitivity (DTH) reactions against Sendai virus. Kinetics of sensitization of effector cells are similar to those described in other virus systems. Maximum DTH response is elicited in the footpad 7 days after sensitization; maximal increase in footpad thickness is found approximately 24 hr after injection of virus and transfer of immune lymphocytes. DTH effector cells are Ig^? and are susceptible to treatment with anti-theta antibody and complement. After transfer of immune lymphocytes a DTH reaction can be elicited by infectious virus, uv light-inactivated virus or cell-cultured Sendai virus which lacks fusion capacity. Requirements of H-2 compatibility are dependent on the biological nature of the virus preparation used for challenge: High doses of infectious or uv light-inactivated Sendai virus require K, D, or IA region compatibility; low concentrations of infectious virus require K and/or D region compatibility, while cell-cultured fusion-negative Sendai virus requires IA region homology. At the level of induction K, D region-restricted DTH-effector T lymphocytes (Td) and cytolytic T lymphocytes (Tc) are stimulated to a greater extent by infectious virus than by uv light-inactivated virus. Furthermore, stimulation of these T lymphocytes is dependent on the route chosen for immunization: intravenous (iv) injection is superior to intraperitoneal (ip) injection; subcutaneous (sc) injection causes the lowest degree of sensitization. IA region-restricted Td lymphocytes are stimulated to comparable amounts by infectious or uv light-inactivated Sendai virus independent of the route of immunization. Td lymphocytes, which require IA region compatibility and Td lymphocytes which require K or D region compatibility can be distinguished by their Lyt phenotype, e.g., IA region-restricted Td lymphocytes are characterized by Lyt-1⁺2^? antigens, while K or D region-restricted Td lymphocytes are phenotypically Lyt-1(⁺)2⁺.     

Thymic lymphocytes: II. Phenotypic modifications of thymocytes after concanavalin A stimulation in the presence of interleukin 2: Early modifications of Lyt 1+2+ subset and later proliferation of cells with more mature phenotypes

Cortical thymocytes are devoid of any immune function, as tested by presently available techniques. The ability of this subpopulation to respond to mitogens or antigens in the presence of interleukin 2 (IL-2) produced by activated mature T lymphocytes has been claimed but is still questioned. In an attempt to study the participation of the different thymocyte subsets and especially that of the cortical type, phenotypic modifications were examined during concanavalin A activation in the presence of IL-2. An immunofluorescent double labeling technique with anti-Lyt 1 and anti-Lyt 2 antibodies was used which led to the determination of four different phenotypes: Lyt 1⁺2⁺, Lyt 1⁺2^?, Lyt 1^?2⁺, and Lyt 1^?2^?. Careful analysis of cell viability in culture and expression of the results in absolute numbers of living cells per culture allowed us to follow modifications of small cellular subsets. Cultures of total thymocytes and PNA-agglutinated (enriched in Lyt 1⁺2⁺ cells) and non-PNA-agglutinated cells (enriched in Lyt 1⁺2^?, Lyt 1^?2⁺, and Lyt 1^?2^? cells) were studied. It was shown that thymocyte activation began by early phenotypic modifications which took place within the first 2 hr of culture but only when Con A plus IL-2 were used. These modifications imply the reduction of the Lyt 1⁺2⁺ pool and a compensatory enhancement of Lyt 1^?2⁺ and Lyt 1^?2^? cells, without modification of the total cell number or [³H]thymidine incorporation. These early phenotypic changes are interpreted as the modulation of antigens on the surface of Lyt 1⁺2⁺ cells. The second phase of thymocyte activation implies cell death (essentially Lyt 1⁺2⁺ cells) and cell proliferation. The cells which specifically proliferate in the presence of Con A and IL-2 are Lyt 1⁺2^? and Lyt 1^?2⁺, the latter always being present in greater number. Cell survival and absolute number of Lyt 1⁺2^? and Lyt 1^?2⁺ cells in the activated PNA^?-enriched population are always higher than in total thymocyte and PNA⁺ cells cultures. Thus, if Lyt 1⁺2⁺ cortical thymocytes do not proliferate by themselves, they seem to intervene by providing Lyt 1^?2⁺ cells which proliferate secondarily.     

Stimulation of splenic T cells on syngeneic monolayers of epidermal basal cells (EBC) in mice. Regulation by Lyt 1+ and Lyt 2+ cell subsets

Syngeneic proliferative response of splenic T cells against monolayers of epidermal basal cells (EBC) was obtained with C57BL/6 and DBA/2 mice. Optimal response, as assessed by [³H]thymidine uptake, occurred on the 6th day of coculture. The level of [^3H]thymidine uptake by unseparated spleen cells was lower than by fractionated T cells from C57BL/6 mice, and null for DBA/2 mice. It was not significantly different when lymphocytes were cocultured with syngeneic or allogeneic epidermal cells. Ia antigens did not appear to be involved in the syngeneic response, since it was not prevented by pretreating stimulator monolayers with monoclonal anti-Ia^k antibody or by adding this antibody directly to the cultures. When the proliferative responses of separated Lyt 1⁺ and Lyt 2⁺ cell subsets were compared, the prominent role of Lyt 1⁺ cells was demonstrated. Enhancement of the T-cell reactivity by eliminating Lyt 2⁺ cells and suppression of the response of a constant number of Lyt 1⁺ cells by adding Lyt 2⁺ cells suggested that Lyt 2⁺ cells could suppress and modulate the Lyt 1⁺ cell proliferation.     

Transplantation tolerance: Evidence for Lyt 1+,2−,Qa 1.2+ suppressor-inducer T cells in allogeneic thymus-grafted nude mice

Nude mice, of BALB/c genotype, grafted with thymus stroma become immunocompetent (R. Hong, H. Schultz-Wisserman, E. Jarreth-Toth, S. D. Horowitz, and D. D. Manning, J. Exp. Med.149, 398, 1979; B. P. Chen and G. A. Splitter, Cell. Immunol.51, 127, 1980), but are tolerant to the thymus-donor genotype. Using such mice to investigate the mechanism(s) of transplantation tolerance, it was found that maintenance of tolerance required active interactions of three subsets of T cells specific for alloantigens of the thymus-donor genotype: (i) Lyt 1⁺,2^? helper T cells, (ii) Lyt 1^?,2⁺ suppressor T cells, and (iii) Lyt 1⁺,2^?,Qa 1.2⁺ suppressor-inducer T cells. In mixed-lymphocyte culture, helper T cells could be activated by alloantigens of the thymus-donor genotype, but clonal expansion of these helper T cells was inhibited by suppressor T cells with the same specificity. Furthermore, exogenous interleukin-2 (IL-2) could modulate this suppressor activity, which suggested that one consequence of suppression was to limit IL-2 available to effector T cells. The response of cultures to exogenous IL-2 also indicated that thymus alloantigen-specific helper T cells had functional IL-2 receptors. Last, the presence of Lyt 1⁺,2^?,Qa 1.2⁺ suppressor-inducer T cells were essential for active suppression, as suppressor T cells could not prevent helper T cells from proliferating to thymus-donor alloantigens when Lyt 1⁺,2^?,Qa 1.2⁺ cells were removed. Altogether, the data presented in this study indicate a feedback-suppression pathway that led to clonal silencing of effector cells in transplantation tolerance.     

Synergism of T lymphocyte subsets in the response to Mls-locus coded antigens during graft-versus-host reaction

After transplantation of lymphoid cells into lethally irradiated (semi)allogeneic mice specific anti-host directed effector T cells are generated. This can be demonstrated using a delayed type hypersensitivity (DTH) assay. In H-2 compatible combinations, Mls-locus antigens, but no other minor histocompatibility antigens, can induce the generation of such effector T cells. This paper shows that maximal anti-host DTH responses are obtained when the lymphoid cells transplanted constitute of a mixture of long-lived, recirculating T2 cells and short-lived, sessile T1 cells. It was demonstrated that anti-Mls locus-directed DTH effector T cells are the progeny of T2 cells, and that T1 cells amplify this response. The latter, however, are by themselves incapable of displaying anti-Mls DTH reactivity. The T1 cells were found to be of the Lyt-1+2+ phenotype, and the T2 cells of the Lyt-1+2- phenotype. The same Lyt phenotypes were found for T1 and T2 cells synergizing in the GvH reaction against H-2 alloantigens.     

Expression of L3T4 molecules on Lyt-2+, class II-restricted antigen-specific T suppressor lymphocytes

Three bovine serum albumin-specific Lyt-2⁺ T suppressor (Ts) cell clones from CBA/J mice have been analyzed with regard to expression of L3T4 molecules. All three Ts-cell clones can be stained with monoclonal antibodies (mAb) to L3T4. Tested for the two clones restricted to recognition of E^k determinants, antigen-specific proliferation on antigen-presenting cells, but not the proliferation induced by conditioned medium can be inhibited by L314-specific mAb. In a similar way, Ts-cell cytolytic effector functions can be blocked by L3T4-specific mAb. Thus L3T4 structures seem to play a role in Ts-cell functions. Furthermore, the data support the view that L3T4 expression can be a property of class II-restricted T cells irrespective of their Lyt phenotype.     

Macrophage procoagulant-inducing activity of influenza-specific effector T cells

Three different types of immune mouse T cells raised against influenza virus were tested for their ability to induce the formation of macrophage procoagulant activity (MPCA) by a macrophage cell line PU5-1.8. They were primary spleen cells, taken 6 days after iv injection of virus, spleen cells from sensitized mice challenged with virus and cultured in vitro for 5 days (secondary cultured cells), and cloned T cells. With the last two preparations, some samples were K,D region restricted, Lyt 2⁺, and had cytotoxic activity; other samples were I region restricted, Lyt 2^?, and were not cytotoxic. Samples of a concanavalin A-activated T-cell supernatant which regularly induced MPCA with PU5-1.8 cells were included as controls in all assays. A few batches of T-cell preparations failed to induce MPCA production, however, most batches were active. Two sources of variation were detected: first, the number of cells (5-to 150-fold) needed to induce a certain level of MPCA, as measured by the decrease in clotting time; and second, the value of the gradient of the cell dose response. Both K,D- and I-region-restricted cells, either as cloned or secondary cultured cells, could induce MPCA but with the latter preparation, I-region-restricted cells were the better inducers by about eightfold. T cells tested in this way were also injected into mouse hind footpads and their ability to mediate delayed-type hypersensitivity (DTH) reactions was measured. A positive but not proportional correlation between the abilities to induce MPCA and mediate DTH activity for primary spleen cells was found, but this was not generally observed with cultured or cloned T cells.     

Expansion of tumor-specific cytolytic T lymphocytes using in vitro restimulation with tumor-specific transplantation antigen

Tumor-specific T lymphocytes (CTL) induced by in vivo immunization of C3H/HeJ mice with the syngeneic methylcholanthrene (MCA)-induced fibrosarcoma MCA-F were expanded in vitro by restimulation with 1-butanol-extracted, isoelectrophoretically purified, tumor-specific transplantation antigen (TSTA) in combination with purified rat interleukin-2 (IL-2) and fresh, syngeneic, 2000-R-irradiated, adherent splenic antigen-presenting cells (APC). The cultured immune T-cell population, containing 40–55% Lyt 2⁺ and 40–60% L3T4⁺ cells, displayed TSTA-specific proliferative and cytotoxic activities in vitro. The expanded T cells appear to recognize butanol-extracted TSTA in association with specific H-2 class I antigens, as revealed by the benefit of syngeneic over allogeneic cells as APC and by the adverse effect of depletion using anti-H-2K, but not anti-Ia, monoclonal antibodies. In adoptive transfer assays in vitro, expanded T cells specifically neutralize homotypic, but not heterotypic, tumor growth in vivo. Based upon the effects of depletion of T-lymphocyte subpopulations using monoclonal antibodies, the Lyt 2⁺ cytotoxic T lymphocytes (CTL) appear to display greater in vivo neutralizing activity than L3T4⁺ T cells. Thus in vitro stimulation of in vivo-immunized T cells, using butanol-extracted TSTA in combination with IL-2 and syngeneic APC, expands tumor-specific CTL.     

Differential responsiveness to MIs locus antigens in graft-versus-host and host-versus-graft reactions

After (semi)allogeneic transplantation of lymphoid cells into lethally irradiated mice, the development of anti-host directed T effector cells can be demonstrated by means of a simple delayed-type hypersensitivity (DTH) assay. Using this assay we have shown that in H-2 compatible combinations Mls locus antigens can induce the generation of such T effector cells during a graft-versus-host (GvH) reaction. Other non-H-2 alloantigens are probably of minor importance. The capacity of Mls locus antigens to induce distinct anti-host DTH reactivity correlated with the capacity to induce a one-way mixed lymphocyte culture (MLC) response. Mls^a and Mls^c locus antigens initiated a positive MLC response as well as distinct GvH-related DTH reactivity. On the other hand, in the combination DBA/2 versus (BALB/c × DBA/2) F₁, the Mls^b locus antigen was not able to initiate in vitro proliferation, a lack of response which coincided with a marginal and short-lasting GvH-related DTH reactivity. In contrast, the host-versus-graft (HvG) DTH reaction of BALB/c and DBA/2 mice to subcutaneously injected (BALB/c × DBA/2) F₁ spleen cells was equally strong. Here antigens other than those coded for by the Mls locus were mainly responsible for the antigraft DTH response. These results suggest that T effector cells generated in GvH and HvG reactions are specific for largely different sets of minor histocompatibility antigens, with a selective stimulation by Mls locus antigens under GvH conditions.     

Restricted recognition of H-2 subregion coded alloantigens in delayed-type hypersensitivity

Subcutaneous (sc) immunization of mice with H-2K, I, or D incompatible spleen cells induces a state of host-versus-graft (HvG) delayed-type hypersensitivity (DTH). The DTH reaction is elicited by challenging the immunized mice in a hind foot with similar allogeneic spleen cells and is measured as the subsequent foot swelling. DTH effector T cells specific for H-2I-coded alloantigens, but not for H-2K/D-coded alloantigens, can be induced in a graft-versus-host (GvH) model as well. In this paper we report that under HvG as well as under GvH conditions the recognition of class II antigens by DTH effector T cells is restricted by class I molecules. Furthermore, DTH effector T cells induced by sc immunization with class I antigens appear to be restricted by class II molecules.     

Generation of nonspecific murine cytotoxic T cells in vitro by purified human interleukin 2

Murine spleen cells developed into nonspecific cytotoxic cells within 72 hr of culture in the presence of highly purified sources of human interleukin 2. In whole spleen cell cultures, human interleukin 2 generated effector cells which were Thy 1.2⁺, Lyt 2.2⁺, resistant to γ irradiation (1000 R), and capable of lysing both H-2 compatible and incompatible targets. The effector cells generated in this manner were not restricted to classical natural killer cell-sensitive targets. If thymus-derived cells (T cells) were depleted from the spleen cell population before culture with human interleukin 2, the effector cells generated were enriched in effectors capable of lysing natural killer cell-sensitive targets. Interferon was not produced in interleukin 2-stimulated spleen cell cultures. In addition, heterologous antibody to murine -γ-interferon did not abrogate the generation of cytotoxic cells by human interleukin 2. These and additional data suggest that human interleukin 2 is capable of stimulating γ-irradiation-sensitive Thy 1.2⁺ cell(s) capable of lysing a variety of target cells regardless of inherent sensitivities to classical natural killer cells. Thy 1.2^? cells were also stimulated by human interleukin 2 and lysed only natural killer cell-sensitive targets. Human interleukin 2 caused some Thy 1.2^? cells to become susceptible to lysis by anti-Thy 1.2 serum and complement.     

In vivo resistance of secondary antitumor immune response to cyclophosphamide: effects on T cell subsets

Summary We have analyzed the effects of high doses of cyclophosphamide (Cy) on primary and secondary antitumor immune response against immunogenic (tum^–) variants of Lewis lung carcinoma (3LL) treated in vitro with UV light. Normal mice and mice previously immunized with tum^– clones were inoculated i.p. with Cy (200 mg/kg body weight) and 24 h later challenged intrafootpad with tum^– or parental 3LL cells. Cy treatment suppressed the primary immune response of normal animals and allowed the growth of tum^– cells. In contrast, Cy-treated immune mice rejected the tumor challenge. The in vivo treatment with Cy decreased the total number of lymphoid cells in the spleens, as well as the proportion of B lymphocytes; however, it increased the percentage of both Lyt2⁺ and L3T4⁺ lymphocytes. Thus, the immunosuppressive effects of Cy on the primary antitumor response could not be attributed to elimination of major T lymphocyte subpopulations. Although the treatment of immune mice with Cy did not significantly impair their antitumor resistance, nor the proportion of Lyt2⁺ and L3T4⁺ lymphocytes in their spleens, the in vitro generation of cytotoxic T lymphocytes (CTL) was markedly reduced.After Cy treatment, the proliferative ability of spleen cells in response to interleukin-2 (IL-2) was substantially impaired. Using monoclonal antibodies to the IL-2 receptor, we found that Cy-treated T lymphocytes failed to fully express the IL-2 receptor following in vitro stimulation with irradiated tumor cells. In line with these findings, the in vitro generation of CTL was not restored by addition of recombinant IL-2 to the cultures. In vivo experiments using purified functional subsets of immune T cells showed that Lyt1⁺, but not Lyt2⁺ lymphocytes were able to transfer antitumor immunity in normal irradiated recipients.Therefore, since Ly1⁺ T lymphocytes were responsible for the antitumor resistance in vivo, the Cy-induced impairment of CTL generation did not affect the ability of immune mice to reject a secondary tumor challenge.This project has been funded at least in part with Federal funds from the Department of Health and Human Services, under contract number NO1-CO-23910 with Resources, Inc. The contents of this publication do not necessarily reflect the view or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government     

Immunotherapeutic potential of blinatumomab-secreting γ9δ2 T Cells

Previous studies have explored the use of engineered blinatumomab-secreting autologous αβ T cells for CD19-targeted cancer therapy. To create a more flexible allogeneic delivery system, we utilized γ9δ2 T cells rather than αβ T cells in a similar application. First, we showed that γ9δ2 T cells could serve as effector cells for blinatumomab, and these effector memory cells could survive for at least 7 days after infusion. The genetically modified blinatumomab-secreting γ9δ2 T cells induced significant cytotoxicity in CD19⁺ tumor cell lines and primary cells from chronic lymphocytic leukemia patients. Of note, blinatumomab-secreting γ9δ2 T cells might also exhibit dual-targeting of CD19 and isopentenyl pyrophosphate, a universal tumor-associated antigen. Furthermore, blinatumomab-secreting γ9δ2 T cells killed CD19-transfected adherent cells, suggesting that the γ9δ2 T cells might be effective for treating solid tumors with appropriate cancer antigens. Together, these results demonstrate the promise of blinatumomab-secreting γ9δ2 T cells as a cancer therapy.     

Human Cytomegalovirus Latency-Associated Proteins Elicit Immune-Suppressive IL-10 Producing CD4+ T Cells

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.     

Characteristics of 2 types of antigen-binding T suppressors formed during an immune response using T-T hybridomas and their extracts

Antigen-binding T cells of mice immunized with low doses of syngenic spleen cells modified by 2,4,6-trinitrophenyl sulphonic acid, were fused with BW 5147.3.13 thymoma subclone. Suppressor hybridomas were not identical in their differentiating antigens and functional activity. Extract from Lyt 1-2+ hybridoma suppressed efferent and afferent limbs of delayed type hypersensitivity (DTH) in recipients sensitized with subcutaneous injection of 3 X 10(7) TNP-SC. Extract from Lyt 1+2+ I-hybridoma suppressed only afferent DTH limb. It is suggested that during DTH induction with low doses of hapten-modified cells the generation of different types of antigen-binding DTH T suppressors takes place.     

Hepatitis B Virus-Infected HepG2hNTCP Cells Serve as a Novel Immunological Tool To Analyze the Antiviral Efficacy of CD8+ T Cells In Vitro

CD8⁺ T cells are the main effector lymphocytes in the control of hepatitis B virus (HBV) infection. However, limitations of model systems, such as low infection rates, restrict mechanistic studies of HBV-specific CD8⁺ T cells. Here, we established a novel immunological cell culture model based on HBV-infected HepG2^hNTCP cells that endogenously processed viral antigens and presented them to HBV-specific CD8⁺ T cells. This induced cytolytic and noncytolytic CD8⁺ T-cell effector functions and reduction of viral loads.     

CD8+ T cells specific for cryptic apoptosis-associated epitopes exacerbate experimental autoimmune encephalomyelitis

The autoimmune immunopathology occurring in multiple sclerosis (MS) is sustained by myelin-specific and -nonspecific CD8⁺ T cells. We have previously shown that, in MS, activated T cells undergoing apoptosis induce a CD8⁺ T cell response directed against antigens that are unveiled during the apoptotic process, namely caspase-cleaved structural proteins such as non-muscle myosin and vimentin. Here, we have explored in vivo the development and the function of the immune responses to cryptic apoptosis-associated epitopes (AEs) in a well-established mouse model of MS, experimental autoimmune encephalomyelitis (EAE), through a combination of immunization approaches, multiparametric flow cytometry, and functional assays. First, we confirmed that this model recapitulated the main findings observed in MS patients, namely that apoptotic T cells and effector/memory AE-specific CD8⁺ T cells accumulate in the central nervous system of mice with EAE, positively correlating with disease severity. Interestingly, we found that AE-specific CD8⁺ T cells were present also in the lymphoid organs of unprimed mice, proliferated under peptide stimulation in vitro, but failed to respond to peptide immunization in vivo, suggesting a physiological control of this response. However, when mice were immunized with AEs along with EAE induction, AE-specific CD8⁺ T cells with an effector/memory phenotype accumulated in the central nervous system, and the disease severity was exacerbated. In conclusion, we demonstrate that AE-specific autoimmunity may contribute to immunopathology in neuroinflammation.Subject terms: Cell death and immune response, Immunological disorders     

Ovarian cancer cytoreduction induces changes in T cell population subsets reducing immunosuppression

Surgery is the primary therapeutic strategy for most solid tumours; however, modern oncology has established that neoplasms are frequently systemic diseases. Being however a local treatment, the mechanisms through which surgery plays its systemic role remain unknown. We have investigated the influence of cytoreduction on the immune system of primary and recurrent ovarian cancer. All ovarian cancer patients show an increase in CD4⁺CD25⁺FOXP3⁺ circulating cells (CD4 T_reg). CD4/CD8 ratio is increased in primary tumours, but not in recurrent neoplasms. Primary cytoreduction is able to increase circulating CD4 and CD8 effector cells and decrease CD4 naïve T cells. CD4⁺ T_reg cells rapidly decreased after primary tumour debulking, while CD8⁺CD25⁺FOXP3⁺ (CD8 T_reg) cells are not detectable in peripheral blood. Similar results on CD4 T_reg were observed with chemical debulking in women subjected to neoadjuvant chemotherapy. CD4 and CD8 T_reg cells are both present in neoplastic tissue. Interleukin (IL)‐10 serum levels decrease after surgery, while no changes are observed in transforming growth factor‐β₁ and IL‐6 levels. Surgically induced reduction of the immunosuppressive environment results in an increased capacity of CD8⁺ T cells to respond to the recall antigens. None of these changes was observed in patients previously subjected to chemotherapy or affected by recurrent disease. In conclusion, we demonstrate in ovarian cancer that primary debulking is associated with a reduction of circulating T_reg and an increase in CD8 T‐cell function. Debulking plays a beneficial systemic effect by reverting immunosuppression and restoring immunological fitness.