Syngeneic mixed lymphocyte reaction in mice: strain distribution, kinetics, participating cells, and absence in NZB mice.

Co-culture of mouse spleen nonadherent (T-enriched cells with mitomycin C-treated unfractionated syngeneic spleen cells resulted in increased DNA synthesis in the responding T cells. The kinetics of this syngeneic mixed lymphocyte reaction (SMLR) showed that peak DNA synthesis occurred on day 5 of culture compared to day 4 for conventional mixed lymphocyte reaction (MLR). Anti-T cell antiserum plus complement treatment of the responding cell population abolished the reaction, and similar treatment of the stimulator population enhanced SMLR. These studies indicate that SMLR represents the response of T cells to non-T cells. Studies on the generation of cytotoxic T lymphocytes (CTL) in parallel cultures of T cells activated by syngeneic or allogeneic spleen cells showed no cytotoxicity of SMLR-activated cells for either PHA- or LPS-induced blasts but did show a good CTL response of allo-activated cells to both targets. Studies on the strain distribution of SMLR revealed that NZB mice manifested poor or no stimulation in SMLR whereas all other strains tested exhibited strong SMLR. This defect in NZB mice may be pathogenetically related to the autoimmune disease that develops in these mice.     

In vivo regulation of the murine syngeneic mixed lymphocyte reaction

Previous work from this laboratory has suggested that a CD8+ T suppressor (Ts) cell network regulated the murine syngeneic mixed lymphocyte reaction (SMLR). We have attempted to disrupt this network by the inoculation of anti-CD8 monoclonal antibodies (mAb) in vivo. Intraperitoneal inoculation of three mAbs resulted in a marked increase in the proliferation of CD4+, self-Ia-reactive splenic T cells in vitro to syngeneic, but not to allogeneic, spleen cells. Suppression was not limited to a specific mouse strain as the enhanced SMLR was reproducible following anti-CD8 treatment of three strains of mice. In vivo depletion of CD8+ T cells was not a prerequisite for enhancement of the SMLR as several mAb to CD8 augmented the SMLR independent of their capacity to cause CD8 T cell depletion. Moreover, enhancement of the SMLR could be mimicked in vitro by inclusion of anti-CD8 mAb in in vitro cultures of responder T cells and irradiated Ia+ syngeneic stimulators. Since the in vitro SMLR was enhanced following mAb treatment, it was expected that the in vivo SMLR would also be increased. However, no evidence of increased in vivo autoreactivity could be detected following in vivo treatment with anti-CD8 mAb, indicating that other mechanisms in addition to CD8+ regulatory T cells acted to regulate the in vivo activity of autoreactive T cells.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

Defective activation of T suppressor cell function in nonobese diabetic mice. Potential relation to cytokine deficiencies

Nonobese diabetic (NOD) is an inbred mouse strain susceptible to development of T cell-mediated autoimmune diabetes. The strain is characterized by high percentages of T lymphocytes in lymphoid organs. The syngeneic mixed lymphocyte reaction (SMLR), a T cell response to self MHC class II Ag, is reportedly involved in the generation of a number of immunoregulatory cells, including suppressor inducers. A severely depressed SMLR characteristic of certain other autoimmune strains was found in NOD but not in nonautoimmune SWR/Bm mice. Moreover, IL-2 produced by NOD T cells at day 6 in an SMLR was at least one hundredfold reduced compared with SWR, and NOD T cells harvested from an SMLR at day 6 were functionally defective when tested for ability to induce suppression of an allogeneic MLR. However, functionally competent suppressor T cells were generated in NOD splenic leukocyte cultures in response to Con A, and IL-2 release from these was equivalent to that released by Con A-stimulated SWR splenocytes. A deficiency in cytokine release was not limited to IL-2, because peritoneal exudate cells from NOD exhibited a greatly diminished sensitivity to LPS-stimulated IL-1 release in comparison to SWR mice. IL-2 supplementation both in vitro and in vivo restored the ability of NOD T cells to respond in a SMLR, with production of cells capable of inducing suppression. Like SMLR-activated T cells from untreated SWR controls, SMLR blasts from IL-2-treated NOD mice were enriched for the L3T4 phenotype. IL-1 supplementation in vitro resulted in partial restoration of T suppressor activation in a SMLR. The depressed SMLR exhibited by NOD mice was apparently a stimulator cell dysfunction, because NOD stimulator cells failed to activate T cells from (SWR x NOD)F1 mice, whereas stimulators from SWR or F1 mice were capable of doing so. Collectively, these results suggest a defect in suppressor cell activation rather than an absence of this immunoregulatory cell population.     

Perturbation of the idiotypic network. II. Transfer of suppression to neonatal mice

The cellular mechanisms which are responsible for idiotype perturbation induced by repeated stimulation with either allogeneic mixed lymphocyte culture-generated syngeneic blasts or allogeneically stimulated syngeneic spleen cells were investigated as described in the preceding article. Using the splenic fragment culture system, the precursor frequencies of T and B cells for anti-phosphorylcholine (PC) antibodies and T15 idiotypic antibodies were determined in allogeneically challenged mice. Adoptive transfers of T cells to neonatal BALB/c mice induced suppression of the T15+ anti-PC responses. In addition, the effect of immunization with internal image-bearing anti-idiotopes on the level of anti-PC antibodies and T15 idiotype was determined. Results from this study demonstrated (i) a decrease in the precursor frequency in the PC-specific and idiotype-specific B cell repertoire; (ii) a decrease in the precursor frequency of T helper cells, which recognize idiotypes and anti-idiotypes; (iii) the possibility to transfer T15 suppression to neonatal mice; and (iv) the possibility to restore T15 dominance by anti-idiotypic antibody immunization. These data indicate that both the T and B compartments are involved in the maintenance of suppression induced by repeated exposure to alloantigen-sensitized syngeneic cells. Collectively these findings show that a nonspecific general suppression induced by allohyperimmunization can perturb the T15+ anti-PC response.     

Enhancement of tumor growth correlates with suppression of the tumor-specific cytolytic T lymphocyte response in mice chronically infected by Trypanosoma cruzi

The immunity of BALB.B mice to syngeneic Gross murine leukemia virus (MuLV)-induced B.GV cells was studied at various times after infection by Trypanosoma cruzi. BALB.B mice chronically infected by the parasite do not develop an effective immune response against B.GV tumor cells, and B.GV tumor growth in vivo is consequently facilitated. The tumor-specific cytolytic T lymphocyte (CTL) compartment in these mice was studied in vitro because CTL are known to participate actively in syngeneic tumor rejection. These analyses showed that: a) CTL differentiation is suppressed in mice with chronic T. cruzi infections; b) suppression is at the level of CTL precursor cell activation; c) suppression is not antigen-specific; and d) suppression is mediated by macrophages and Lyt-2+ T lymphocytes.     

Tolerance induced by TNP-derivatized syngeneic erythrocytes: evidence for cooperation between hapten-specific T and hapten-specific B lymphocytes in the immune response.

Tolerance to the DNP haptenic determinant was induced with a single i.v. injection of trinitrophenylated syngeneic red blood cells. The tolerant state lasted 1 month and was stable on transfer to irradiated thymectomized syngeneic recipients. Suppressor activity was found soon after injection of tolerogen but was lost before the termination of tolerance. The unresponsive state could be reversed by adding normal thymus cells to tolerant spleen cells but not by normal bone marrow cells. LPS when given with immunogen restored the normal immune response in tolerant mice. Thus the injection of TNP-MRBC induced partial immune unresponsiveness which was characterized by the induction of T cell suppressor activity and by a hapten-specific helper T cells tolerance. Finally, these studies suggest a cooperative interaction between DNP-specific T lymphocytes and DNP-specific B lymphocytes in the immune response to DNP-BGG.     

T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. III. Dissociation of primed cytolytic T-cell and efferent suppressor-T-cell activity following intravenous injection of trinitrophenol-modified syngeneic spleen cells

The parenteral injection of ligand-coupled syngeneic spleen cells has profound effects on immune responsiveness. In this regard, it was examined whether the primed in vitro trinitrophenol (TNP)-specific cytotoxic T-lymphocyte (CTL) responses observed in splenic T-cell populations from mice injected intravenously (iv) with syngeneic TNP-modified spleen cells (TNP-SC) are related to the efferent-acting suppressor-T-cell (Ts) activity observed in splenocytes from iv primed mice. Treatment of mice with cyclophosphamide, adult thymectomy, or monoclonal anti-I-J antiserum prior to the iv injection of TNP-SC was found to eliminate the ability of splenic Ts from these mice to suppress the passive transfer of delayed-type hypersensitivity (DTH) mediated by trinitrochlorobenzene-immune T cells. In contrast, spleen cells from these pretreated mice showed no impairment in the development of augmented TNP-specific CTL responses upon in vitro restimulation with TNP-SC. Separation of the two activities was also achieved in a kinetic analysis. It is concluded that specific enhancement of CTL responsiveness induced by the iv injection of TNP-SC is related to the expansion of a population prelytic Lyt 2+ CTL effector cells which does not appear to contain efferent-acting Lyt 2+ Ts active in suppressing DTH expression.     

Immunoregulatory abnormalities in autoimmune thyroid diseases

We have investigated the ability of lymphocytes from normal subjects and patients with autoimmune thyroid diseases to respond to a thyroidal antigen (human thyroglobulin, hTG) and a non-thyroidal antigen (Keyhole limpet hemocyanin, KLH) in vitro, using a hapten (trinitrophenol, TNP)-carrier system. This system is based on the concept that the T helper cells which respond to hTG or KLH should stimulate anti-TNP antibody producing B cells in the presence of TNP conjugated hTG (TNP-hTG) or KLH (TNP-KLH). After 5 or 6 days of culture of peripheral blood mononuclear cells with pokeweed mitogen (PWM), PWM and TNP-hTG, or PWM and TNP-KLH, IgM anti-TNP and IgM anti-sheep red blood cell (SRBC) plaque forming cells (PFC) were enumerated. The results showed that (1) in normal controls, hTG caused only suppression in both TNP and SRBC response, and KLH caused dose-related enhancement and suppression in TNP response without a change in SRBC response, and (2) in patients, both hTG and KLH resulted in dose-related enhancement in TNP response without a change in SRBC response. These data suggest that patients with autoimmune thyroid diseases have regulatory cell abnormalities confined to a thyroid antigen.     

Clonal anergy of memory B cells in epitope-specific regulation.

Immunization of carrier (keyhole limpet hemocyanin (KLH)) primed mice with the hapten-carrier TNP-KLH induces specific suppression for the IgG anti-TNP-response without interfering with the response to epitopes on the carrier molecule. To examine the status of hapten-specific memory B cells from suppressed mice, highly enriched populations of TNP-specific memory B cells were purified from the spleen of TNP-KLH (control) or KLH/TNP-KLH (suppressed) immunized mice and tested in vitro for their ability to respond to TD or TI (TNP-KLH, TNP-LPS) antigenic challenge in presence of a KLH-specific Th cell line. Similar numbers of TNP-specific B cells with the characteristics of memory B cells were obtained from control and suppressed mice. TNP-specific B cells from suppressed mice could be triggered to IgG production by TNP-LPS but had an impaired ability to differentiate into IgG-secreting cells in response to TNP-KLH. This impaired IgG response to TNP-KLH was not due to an active suppression by a subset of TNP-specific B cells, or to an impedence of memory cells to a class switching but to an intrinsic memory B cell defect. TNP-specific B cells from suppressed mice were as efficient as memory B cells from control mice to present TNP-KLH to KLH-specific Th cells and to proliferate in response to T cell help. Our data support the view that the effector mechanism of epitope specific regulation does not interfere with the development of hapten-specific memory B cells but that these cells have an intrinsic defect that prevents their differentiation into active IgG antibody secreting cells in response to a T-dependent antigenic challenge.     

Production of specific macrophage-arming factor precedes cytotoxic T lymphocyte activity in vivo during tumor rejection

Summary Recently we published a hypothesis on the immunological events occurring during tumor rejection. One of the implications of this hypothesis is that specific macrophage-arming factor (SMAF) is produced early during the initiation of the immune response, whereas the classical cell-mediated immune response components, such as cytotoxic T lymphocytes (CTL), are produced later, that is, during the amplifier-effector phase. In this paper we establish the kinetics of the induction of (a) lymphocytes producing SMAF and (b) CTL. Groups of DBA/2 mice were injected i.p. once, twice or three times with irradiated and/or non-irradiated syngeneic SL2 tumor cells, the injections being given at intervals of 10 days. After each of these injections the production of SMAF and the expression of CTL activity were established. The results showed that in the peritoneal cavity SMAF-producing lymphocytes appeared earlier than cytotoxic lymphocytes (CTL). In addition, it was shown (a) that SMAF does not interfere with the in vitro cytotoxicity expressed by CTL and (b) that in addition to CTL memory cells, SMAF-producing memory cells were also induced after injection of syngeneic tumor cells. These data support the hypothesis that SMAF is involved in the early phase of the cellular immune response against tumors, whereas CTL are induced later.     

In vitro reactivity of splenic lymphocytes from normal and UV-irradiated mice against syngeneic UV-induced tumors.

Skin tumors induced in mice by chronic ultraviolet (UV) irradiation are highly antigenic and are frequently immunologically rejected upon transplantation to normal syngeneic recipients. In this study we characterized this immune response with an in vitro microcytotoxicity test. Cytotoxic activity was present in the spleen cells of mice given a single injection of syngeneic UV-induced fibrosarcoma cells. After removal of adherent spleen cells, the remaining splenic lymphocytes were specifically cytotoxic for the immunizing tumor and showed no cross-reactivity with other syngeneic UV-induced or methylcholanthrene-induced tumors of similar histologic type. The level of cell-mediated reactivity against UV-induced tumors was quite high compared to that obtained with syngeneic tumors induced by methylcholanthrene, and the cytotoxicity was attributable to a population of theta antigen-bearing lymphocytes. With this in vitro test, we compared the response of normal mice, which reject a syngeneic tumor challenge, with that of UV-irradiated mice, in which the syngeneic UV-induced tumors grow progressively. After tumor cell inoculation, lymphocytes form the unirradiated (regressor) mice showed a high degree of cytotoxicity that reached a maximum level 8 days after injection. In contrast, no reactivity could be detected in the spleens of tumor-challenged UV-irradiated (progressor) mice.     

Effect of trypsin on suppressor cell formation and function in localized staphylococcal infection

The present study has been made on (CBA X C57BL)F1 mice immunized with sheep red blood cells (SRBC) and inoculated with staphylococci (M-SRBC-S). The injection of splenic lymphocytes from syngeneic M-SRBC-S into intact mice has been found to suppress immune response to SRBC in these mice. The injection of trypsin into M-SRBC-S decreases the suppressive action of their lymphocytes on SRBC-induced immune response in syngeneic recipients. The injection of trypsin into the recipients has been found to produce no effect on the immunosuppressive action of transplanted lymphocytes obtained from M-SRBC-S. The injection of trypsin into M-SRBC-S induces the release of the factor, inhibiting the formation and function of suppressor cells, by their splenocytes. Previously formed suppressor cells block the release of the immunostimulating factor by the splenocytes of the animals receiving the injections of trypsin.     

Surface phenotype of responder cells in the syngeneic mixed lymphocyte reaction in mice

The organ distribution and surface phenotype of SMLR responder cells has been investigated. Nylon-wool-passed spleen cells, which proliferate in response to mitomycin-C-treated syngeneic spleen cells, are Thy 1.2⁺ Ly 1⁺2^?3^?. SMLR responder cells are not confined to the spleen since nylon-wool-nonadherent lymph node cells as well as unfractionated thoracic duct lymphocytes show activity. Responder cells have characteristics of mature T cells since cortisone-resistant thymocytes, but not thymocytes from untreated mice, are capable of SMLR response. In addition, naturally occurring thymocytotoxic antibody (NTA), which in our experiments exhibits cytotoxicity only for thymocytes, does not appear to affect the subpopulation of the T cells which respond in the SMLR.     

B7-deficient autoreactive T cells are highly susceptible to suppression by CD4(+)CD25(+) regulatory T cells

CD4(+)CD25(+) regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4(+)CD25(+) T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.     

T cell regulation of B cell activation. An antigen-mediated tripartite interaction of Ts cells, Th cells, and B cells is required for suppression

To determine the requirements underlying the antigen specificity observed in T cell-mediated immune response suppression, cloned major histocompatibility complex (MHC)-restricted T suppressor (Ts) cells specific for keyhole limpet hemocyanin (KLH) and cloned MHC-restricted T helper (Th) cells specific for fowl gamma-globulin (FGG) were employed to study the regulation of trinitrophenyl (TNP)-specific B cell responses. Neither antigen bridging between Ts cells and Th cells (FGG=KLH) nor bridging between Ts cells and B cells (TNP-KLH) was sufficient to allow suppression; a mixture of FGG=KLH and TNP-KLH was also insufficient for suppression. In contrast, suppression was induced by KLH-specific Ts cells only when suppressor determinants (KLH), helper determinants (FGG), and B cell determinants (TNP) were covalently linked on the same molecule (TMP-FGG)=(TNP-KLH) or TNP-(FGG=KLH)). These findings imply that a tripartite antigen-mediated interaction of Ts cells, Th cells, and responding B cells is necessary for the mediation of this antigen-specific suppression.     

Modulation of Immune Response by Bacterial Lipopolysaccharide (LPS): Roles of Macrophages and T Cells in In Vitro Adjuvant Effect of LPS on Antibody Response to T Cell-Dependent and T Cell-Independent Antigens

The roles of macrophages and T cells in the adjuvant effect of lipopolysaccharide (LPS) were studied. In vitro anti-trinitrophenyl (anti-TNP) antibody responses to TNP-Ficoll and TNP-keyhole limpet hemocyanine (TNP-KLH) in spleen cells of C57BL/6 mice showed the most enhancement, when LPS was added to cultures at 1 μg/ml 48 hr after culture was started. The responses to these antigens were enhanced markedly by LPS in whole and macrophage-depleted spleen cells. The enhancement was greater in the latter group than in the former. The adjuvant effect among whole, T cell-depleted, macrophage-depleted and both macrophage- and T cell-depleted spleen cells was compared. The response to TNP-Ficoll was enhanced markedly by LPS in all groups. The enhancement was greater in the latter two groups than in the first two groups. The response to TNP-KLH was enhanced by LPS strongly in macrophage-depleted spleen cells, moderately in whole and both macrophage- and T cell-depleted spleen cells, and only slightly in T cell-depleted spleen cells. Enhancement was restored to T cell-depleted spleen cells by adding T cells. The response to TNP-KLH of macrophage-depleted spleen cells of LPS-responsive C3H/HeN mice which was enhanced by LPS was suppressed by adding splenic macrophages of C3H/HeN mice, but not of LPS-nonresponsive C3H/HeJ mice. The response to TNP-KLH of macrophage-depleted spleen cells of C3H/HeJ mice was not enhanced by LPS, irrespective of the addition of macrophages of C3H/HeN mice. The results indicate that B cells are activated directly by LPS, and T cells enhance and macrophages suppress the adjuvant effect of LPS.     

The regulatory role of NZB T lymphocytes in the production of anti-DNA antibodies in vitro

Murine lupus is characterized by the production of numerous autoantibodies and immune complex glomerulonephritis. Anti-DNA antibodies are the hallmark of this disorder and may be associated pathogenetically with the glomerulonephritis. The cellular mechanisms underlying the regulation of the production of anti-DNA antibodies may prove to be the fundamental abnormalities responsible for the lupus syndrome seen in these mice. By using a system of spontaneous anti-DNA antibody production in vitro, we have determined that such production is characteristic of autoimmune NZB and MRL-lpr/lpr mice but not of the nonautoimmune control strains. Additional examination of the cellular mechanisms involved in the regulation of this response in NZB mice revealed: 1) this response is markedly T cell dependent, 2) NZB T cells are essential for maximal production of this autoantibody, and 3) NZB T cells actively interfere with normal immune regulatory mechanisms that lead to the production of anti-DNA antibodies spontaneously in vitro by nonautoimmune syngeneic B lymphocytes. Although these studies of anti-DNA antibody production in vitro disagree with previous work by others they successfully reproduce the results obtained earlier in experiments performed in vivo.     

Extracellular nicotinamide adenine dinucleotide induces t cell apoptosis in vivo and in vitro.

Incubation of mouse T cells expressing the cell surface enzyme ADP ribosyltransferase with nicotinamide adenine dinucleotide (NAD) had been reported to cause ADP ribosylation of cell surface molecules, inhibition of transmembrane signaling, and suppression of immune responses. In this study, we analyze the reasons for these effects and report that contact of T cells with NAD causes cell death. Naive T cells when incubated with NAD and adoptively transferred into semiallogeneic mice fail to cause graft-vs-host disease, and when injected into syngeneic, T cell-deficient recipients do not reconstitute these mice. Rather, they accumulate in the liver, leading to an increase of apoptotic lymphocytes in this organ. Similar effects are induced by injection of NAD, shown to cause a dramatic increase of apoptotic CD3(+), CD4(+), and CD8(+) cells in the liver. Consistent with this, in vitro incubation of naive T cells with NAD is shown to induce apoptosis. In contrast, no cell death is demonstrable when T cells are activated before incubation with NAD. It is concluded that ecto-NAD, as substrate of ADP ribosyltransferase, acts on naive, but not on activated CD69(+) T cells.     

Peritumoral CpG DNA elicits a coordinated response of CD8 T cells and innate effectors to cure established tumors in a murine colon carcinoma model

The immune system of vertebrates is able to detect bacterial DNA based on the presence of unmethylated CpG motifs. We examined the therapeutic potential of oligodeoxynucleotides with CpG motifs (CpG ODN) in a colon carcinoma model in BALB/c mice. Tumors were induced by s.c. injection of syngeneic C26 cells or Renca kidney cancer cells as a control. Injection of CpG ODN alone or in combination with irradiated tumor cells did not protect mice against subsequent tumor challenge. In contrast, weekly injections of CpG ODN into the margin of already established tumors resulted in regression of tumors and complete cure of mice. The injection site was critical, since injection of CpG ODN at distant sites was not effective. Mice with two bilateral C26 tumors rejected both tumors upon peritumoral injection of one tumor, indicating the development of a systemic immune response. The tumor specificity of the immune response was demonstrated in mice bearing a C26 tumor and a Renca tumor at the same time. Mice that rejected a tumor upon peritumoral CpG treatment remained tumor free and were protected against rechallenge with the same tumor cells, but not with the other tumor, demonstrating long term memory. Tumor-specific CD8 T cells as well as innate effector cells contributed to the antitumor activity of treatment. In conclusion, peritumoral CpG ODN monotherapy elicits a strong CD8 T cell response and innate effector mechanisms that seem to act in concert to overcome unresponsiveness of the immune system toward a growing tumor.