Tumor dormancy. I. Regression of BCL1 tumor and induction of a dormant tumor state in mice chimeric at the major histocompatibility complex

The growth of the BCL1 tumor in murine H-2 chimeras was studied. Lethally x-irradiated BALB/c mice were reconstituted with C57BL/6 bone marrow that had been depleted of T cells. When chimerism was established 90 to 120 days later, large doses of BCL1 cells were injected. The tumor grew progressively, reaching a peak level of as many as 10(9) tumor cells per animal by 40 days after inoculation. After that time, the tumor regressed in all the chimeric animals, and by 100 days after inoculation, virtually all the animals appeared disease free as judged by an absence of BCL1-idiotype-positive cells in the spleen and peripheral blood, a normal spleen size, and absence of an elevated white blood cell count. Such animals were followed for as long as 8 mo after tumor inoculation and remained disease free. However, transfer of graded numbers of splenocytes from these animals into normal BALB/c recipients resulted in development of tumor in recipients receiving 100 or more spleen cells. These results indicate a large tumor burden in the spleen of each donor, namely, 10(6) to 10(7) BCL1 cells. The present model should facilitate characterization of the mechanisms underlying tumor dormancy.     

Influence of age,castration, and testosterone on T cell subsets in healthy and leukemia grafted mice

The distribution of T cell subsets in pubertal (2 months) and post-pubertal (10 months) mice showed a significant decrease in the percentage of CD4+ splenocytes and peripheral blood lymphocytes (PBL) with age, unlike the percentage of CD8+ cells in PBL, which remained unchanged. The change in the distribution of T cell subsets in the spleen and blood occurred in 2 months old castrated mice, as in 10 months old animals. P388 tumor grew better in post-pubertal and in castrated mice than in young mice. The intact mice survived longer than the castrated ones. The relative number of CD4+, CD8+ and CD2+ splenocytes was lower in transplanted intact mice than that in controls. The CD8+ and CD2+ subsets in the blood of 2 months transplanted mice were higher than those in controls, whereas in PBL, in 10 months old and castrated mice, the T lymphocyte subsets remain unchanged. Depo-testosterone (DT) injection strongly reduced weight and tumor growth in all the intact and castrated animals. A significant correlation is observed between the tumor weight and testosterone level in the plasma of the 2 months old DT treated mice. Moreover, DT injection induced a significant increase in the percentage of blood CD8+ cells in all the batches. These data indicate that physiologically, androgens affect the age-related distribution of lymphocyte T subsets and suggest that they slow down tumor growth, besides causing a direct effect, through an immunological process.     

A model of Ph' positive chronic myeloid leukemia-blast crisis cell line growth in immunodeficient SCID mice.

A human Philadelphia-chromosome positive chronic myeloid leukemia-blast crisis (CML-BC) cell line BV173 proliferated in the hematopoietic tissues, infiltrated various organs and caused the death of immunodeficient SCID mice. Leukemia spreading was assessed with diminished number of bone marrow cells and caused splenomegaly. The leukemic colonies grew from single cell suspension of bone marrow, spleen and peripheral blood. Bcr-abl m-RNA was detectable in bone marrow, spleen, peripheral blood, liver, lungs and brain. Dying mice demonstrated severely hypoplastic bone marrow, splenomegaly and massive metastases in the liver and kidneys. The survival time of animals was dependent on the number of inoculated leukemia cells.     

Chronic alcohol consumption impairs distribution and compromises circulation of B cells in B16BL6 melanoma-bearing mice

Accumulating research indicates that B cells are involved in anti-tumor immunity. Chronic alcohol consumption is associated with decreased survival of cancer patients. The effect of alcohol consumption on B cells in tumor-bearing hosts is unknown. Results in melanoma-bearing mice showed that chronic alcohol consumption did not alter the percentage and number of B cells in bone marrow, spleen, and lymph nodes but dramatically decreased B cells in the peripheral blood. Alcohol consumption did not alter the development of B cells in the bone marrow and did not affect follicular B cells in the spleen; however, it increased T1 B cells and decreased marginal zone B cells in the spleen. Alcohol consumption also decreased mature B cells in the blood. It did not alter the chemotactic capacity of plasma to facilitate migration of splenocytes or the chemotactic response of splenocytes to CXCL13 and CCL21. However, the response of splenocytes to sphingosine-1-phosphate was impaired in alcohol-consuming, melanoma-bearing mice. The expression of sphingosine-1-phosphate receptor-1 (S1PR1) and sphingosine-1-phosphate lyase-1 (SPL1) in splenocytes was downregulated. Taken together, these results indicate that chronic alcohol consumption decreases peripheral blood B cells by compromising B cell egress from the spleen. The downregulation of S1PR1 and SPL1 expression in alcohol-consuming, melanoma-bearing mice could be associated with compromised egress of B cells from the spleen.     

Abrogation of the capacity of delayed-type hypersensitivity responses to alloantigens by intravenous injection of neuraminidase-treated allogeneic cells

BALB/c or C3H/He mice were inoculated i.v. with allogeneic spleen cells untreated or treated with neuraminidase. Appreciable or potent anti-allo-delayed-type hypersensitivity (DTH) responses were observed when mice were inoculated i.v. with untreated allogeneic cells or inoculated i.v. with those cells followed by s.c. immunization with untreated allogeneic cells. In contrast, i.v. inoculation of neuraminidase-treated allogeneic cells (presensitization) not only failed to induce any significant anti-allo-DTH responses but also abolished the capability of the animals to develop DTH responses after s.c. immunization, indicating the tolerance induction. This tolerance was alloantigen-specific, and rapidly inducible and long lasting. The induction of suppressor cell activity was demonstrated in tolerant mice. However, this activity was associated only with the tolerant state around 4 to 7 days after the i.v. presensitization, but was no longer detected in mice more than 14 days after the presensitization, although these mice exhibited complete tolerant state. When spleen cells from such tolerant mice were transferred i.v. into 600 R x-irradiated syngeneic recipient mice alone or together with normal syngeneic spleen cells, these tolerant spleen cells themselves failed to induce DTH responses but did not exhibit suppressive effect on the generation of DTH responses induced by normal spleen cells co-transferred. These results indicate that i.v. administration of neuraminidase-treated allogeneic cells results in the induction of alloantigen-specific tolerance which is not always associated with the induction of suppressor cell activity but rather with the elimination or functional impairment of alloantigen-specific clones.     

Chimeric drift in allophenic mice.

The composition of the immune system of 33 allophenic mice of four different types [C57BL/6 in equilibrium DBA/1, C57BL/6 in equilibrium (CBA X CBA/H-T6), C57BL/6 in equilibrium (A X SJL), DBA/1 in equilibrium (CBA X CBA/H-T6)] was studied. It was found that the parental composition of the peripheral white blood cells changed significantly during a two-month interval in 11/33 or 33% of the mice studied. This phenomenon has been termed chimeric drift. The animals were sacrificed between 9 and 16 months of age, and the parental composition of the peripheral white blood cells, spleen white blood cells, and thymocytes was determined on the day of sacrifice. It was foound that the peripheral white blood cell and spleen white blood cell compositions showed a high degree of correlation. However 8/33 or 24% of the mice studied showed discordance of the spleen and thymocyte cell populations. Seven of the 8 mice which showed spleen-thymocyte discordance, had also shown evidence of chimeric drift earlier in their lives. We suggest that this is evidence that chimeric drift may have an immunological basis.     

Studies on the induction of tolerance to alloantigens. I. The abrogation of potentials for delayed-type-hypersensitivity response to alloantigens by portal venous inoculation with allogeneic cells

The present study investigates the effect of portal venous (p.v.) administration of allogeneic cells on the capacity of delayed-type-hypersensitivity (DTH) reactivity to alloantigens. BALB/c mice were inoculated with C3H/He spleen cells via intravenous (i.v.) or p.v. route. Intravenous injection of C3H/He spleen cells into BALB/c mice resulted in appreciable DTH responses to C3H/He alloantigens. In contrast, p.v. inoculation of the same number of C3H/He cells not only failed to induce any significant anti-C3H/He DTH responses but also abolished the capability of the animals to develop DTH responses as induced by subcutaneous (s.c.) immunization with C3H/He spleen cells. Such suppression was alloantigen-specific, since p.v. inoculation of C3H/He spleen cells resulted in selective inhibition of anti-C3H/He DTH potential without suppressing DTH responses to C57BL/6 alloantigens. This tolerance was rapidly inducible and long-lasting. When spleen cells from tolerant mice were transferred i.v. into 600 R X-irradiated syngeneic recipient mice alone or together with normal BALB/c spleen cells, these tolerant spleen cells themselves failed to induce DTH responses but did not exhibit any suppressive effect on the generation of DTH responses induced by normal spleen cells co-transferred. These results indicate that tolerance was not necessarily associated with the induction of suppressor cell activity but rather was associated with the elimination or functional impairment of clones specific for alloantigens. The results are discussed in the context of a) the role of the liver in immune responses, b) cellular mechanisms underlying the tolerance induction, and c) potential application of this approach to the future transplantation immunology.     

Effects of a murine mammary tumor on in vivo and in vitro hemopoiesis

The effects of an autologous transplanted mammary tumor (RIII-T3) on hemopoiesis in RIII mice are described. Tumor-bearing animals died 30 to 40 days after inoculation and displayed splenomegaly, extreme neutrophilia, and moderately increased monocyte levels in the spleen, peripheral blood, and bone marrow. The precursors of neutrophils and monocytes, granulocyte/macrophage colony-forming cells (GM-CFC) were elevated in the spleen, bone marrow, and peripheral blood. RIII-T3-conditioned medium stimulated bone marrow GM-CFC and caused the myelomonocytic cell line, WEHI-3B, to differentiate in vitro. The conditioned medium did not stimulate erythroid, megakaryocyte, or eosinophil colony formation. When conditioned medium was fractionated, two peaks of activity corresponding to GM-CSF and G-CSF were observed, suggesting that the extreme neutrophilia observed in tumor-bearing animals may result from chronic exposure of the hemopoietic system to these hemopoietic hormones.     

Immunodepressed Status of Mice after Bulbectomy

We studied the immune response in lymphoid cells of mice subjected to bilateral olfactory bulbectomy in comparison with sham-operated animals 1.5 and 13 months after surgery. The concentration of tumor necrosis factor decreased threefold in the peripheral blood of bulbectomized mice 1.5 months after surgery. Signs of immunodepression were also observed 13 months after surgery: suppression of mitogen-stimulated proliferation of T and B lymphocytes in the spleen, inhibition of synthesis of tumor necrosis factor in peritoneal macrophages and splenocytes, and decreased macrophage NO production. According to the immune status indices and our previous data on behavioral, biochemical, and morphological changes induced in bulbectomized mice, they have common symptoms with the Alzheimer's disease. This allows us to consider such animals as a model of sporadic form of this disease rather than of a depression.     

Effect of the expression of a hepatocyte-specific MHC molecule in transgenic mice on T cell tolerance

A hybrid murine class I gene, Q10/L, was injected into C3H/HeJ fertilized ova to produce transgenic (TG) mice. This fusion gene contained 414 bp of Q10 promoter sequences which was sufficient to direct liver-specific expression in two lines of animals. Animals from these lines did not have Q10/L mRNA in 10 nonhepatic tissues examined including thymus, spleen, and bone marrow. The ontogeny of Q10/Ld expression in both liver and yolk sac paralleled expression of endogenous Q10. Analysis of liver cells from these lines by flow cytometry and immunofluorescence demonstrated the presence of the Q10/L Ag solely on hepatocytes. TG animals showed no signs of hepatic disease as evidenced by an absence of cellular infiltrates in the liver and a normal profile of serum enzymes that are elevated in association with hepatic disease. When spleen cells from TG animals were cocultured with splenocytes that express Ag cross-reactive with Q10/L, CTL were generated that recognized and lysed L cells which express Q10/L. However, the extent of lysis was less than that generated from non-TG control littermates. That these cross-reactive T cells were physiologically significant was demonstrated by adoptive transfer of in vivo primed T cell enriched spleen cells which produced a mononuclear infiltration of the liver of TG recipients. However, inoculation of Q10/L L cells or splenocytes expressing Q10/L cross-reactive Ag into TG mice did not induce cellular infiltration or overt hepatic disease. Whereas inoculation of normal C3H mice with these cells led to priming of Q10/L reactive CTL, anti-Q10/L CTL could not be primed in TG mice. This suggests that Ag expression solely on hepatocytes can lead to inactivation of specific CTL clones and thus account for the observed in vivo tolerance.     

Cultivation of murine B-cell hybridomas in the spleen

The tumorigenic capacity of mouse B-cell hybridomas in both cloned and primary cultures was studied. The cells were selected for inoculation from 24-well plates and introduced into the spleen of syngeneic mice. The cells took in 50% of the animals. The cells of hybridoma tumors induced as the result of intrasplenic inoculation, when subcultured in the second passage following the standard scheme, i.e. inoculated intraperitoneally in a dose of 1 X 10(7) cells into mice previously treated with vaseline oil or pristane, produced tumors in 100% of the animals.     

Pathogenicity of molecularly cloned bovine leukemia virus.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.     

Measles Virus Spread and Pathogenesis in Genetically Modified Mice

Attenuated Edmonston measles virus (MV-Edm) is not pathogenic in standard mice. We show here that MV-Edm inoculated via the natural respiratory route has a limited propagation in the lungs of mice with a targeted mutation inactivating the alpha/beta interferon receptor. A high dose of MV-Edm administered intracerebrally is lethal for about half of these mice. To study the consequences of the availability of a high-affinity receptor for MV propagation, we generated alpha/beta interferon-defective mice expressing human CD46 with human-like tissue specificity. Intranasal infection of these mice with MV-Edm resulted in enhanced spread to the lungs and more prominent inflammatory response. Virus replication was also detected in peripheral blood mononuclear cells, the spleen, and the liver. Moreover, intracerebral inoculation of adult animals with low MV-Edm doses caused encephalitis with almost inevitably lethal outcome. We conclude that in mice alpha/beta interferon controls MV infection and that a high-affinity receptor facilitates, but is not strictly required for, MV spread and pathogenesis.     

Down-regulation of natural killer cell activity in MoLV leukemogenesis: evidence for tumor-cell-mediated suppression

The role of natural killer (NK) cells in retrovirus-induced leukemogenesis was studied. These cells which do not require prior sensitization are considered as a part of the body's defense system against tumor development and spread. Neonate BALB/c mice infected with Moloney murine leukemia virus (MoLV) develop leukemia within 3-6 months. The MoLV-infected mice showed a progressive loss of endogenous and augmented NK activity, correlated with the development of the leukemic state. Mixing of spleen cells from tumor-bearing mice with NK-augmented splenocytes resulted in suppression of NK activity. In addition, mixing of T cell lines isolated from MoLV-induced tumors with augmented splenocytes also resulted in the down-regulation of NK cell activity. The present study demonstrates that tumor cells from leukemic organs and leukemic T cell lines can actively suppress NK cell function. It is postulated that after MoLV infection the progression of virus-transformed T cells to a fully developed tumor depends on the ability of these cells to down-regulate NK cell activity and thus escape immune surveillance.     

Lyt-2+ T cell-mediated protection against listeriosis. Protection correlates with phagocyte depletion but not with IFN-gamma production

The transfer of listeria-immune splenocytes into non-immune mice markedly increases host resistance to listeriosis. To study the mechanism for this enhancement, we compared the inflammatory response to infection in nonimmune and adoptively immunized mice. Despite much better containment of bacterial growth, adoptively immunized animals accumulated significantly fewer phagocytes (neutrophils and macrophages) in the spleen and liver than controls. Immune T cells not only inhibited phagocyte accumulation but also reduced the in vitro anti-listerial activity of splenocytes. Significant differences in phagocyte accumulation were observed even when the initial listeria dose was adjusted to produce comparable spleen listeria loads in immune and non-immune animals. However, bone marrow and peripheral blood phagocyte counts were similar in both groups. Depletion of Lyt-2+ cells (using mAb and C) from donor splenocytes prevented the transfer of protection and increased phagocyte accumulation without altering listeria-dependent IFN-gamma production by donor or recipient splenocytes in vitro. L3T4 depletion did not affect host resistance or phagocyte accumulation even though it reduced in vitro interferon production by donor cells. Hence the different effects of L3T4+ and Lyt-2+ cells in vivo cannot be explained simply by variations in IFN production. We suggest this paradoxical suppression of phagocyte accumulation during adoptive transfer may reflect lysis of bacteria-laden phagocytes by listeria-specific Lyt-2+ cells in vivo. Selective destruction of older, heavily infected cells might contribute to host resistance by eliminating a potential site for intracellular proliferation of bacteria.     

Depletion of CD4+ (L3T4+) lymphocytes in vivo impairs murine host defense to Cryptococcus neoformans

T cell-mediated immunity has been shown to play an important role in the host defense to Cryptococcus neoformans. Infections due to C. neoformans are increased in patients with AIDS who are deficient in the CD4+ subset of T lymphocytes. Thus, the effect of CD4+ (L3T4+) lymphocyte depletion on murine host defenses to C. neoformans was studied. The mAb GK 1.5 was administered to mice, and CD4+ T lymphocyte depletion was confirmed by the analysis of T cell subsets in blood, spleen, lymph node, and lung. Evidence of a functional defect was confirmed by demonstrating that the splenocytes of treated mice were unable to proliferate in response to class II incompatible spleen cells. Furthermore, delayed type hypersensitivity to C. neoformans was abrogated by CD4+ lymphocyte depletion. Mice depleted of CD4+ lymphocytes were inoculated with a virulent strain of C. neoformans by the i.v. or the intratracheal route. After i.v. inoculation of C. neoformans, the survival of mice depleted of CD4+ lymphocytes was reduced (27.8 +/- 1.8 vs 36.0 +/- 3.1 days, p less than 0.04). After intratracheal inoculation, C. neoformans disseminated from the lung to extrapulmonary organs. Dissemination occurred earlier in mice depleted of CD4+ lymphocytes compared to mice that received control antibody, and the burden of C. neoformans in extrapulmonary organs was greater in mice depleted of CD4+ lymphocytes than control mice. Surprisingly, there was no increase in the burden of C. neoformans in the lungs of CD4+ lymphocyte-depleted mice. Survival of mice inoculated with C. neoformans and depleted of CD4+ lymphocytes was reduced compared to control mice and was related to the increased rate of accumulation of organisms in the brains of treated mice. The mean survival of GK 1.5-treated mice was 34.1 +/- 0.9 days compared to control mice with a mean survival of 40.6 +/- 9 days (p less than 0.001). These data suggest that CD4+ lymphocytes play a prominent role in the host defense of infections due to C. neoformans, that CD4+ lymphocytes are required in extrapulmonary organs for optimal clearance of C. neoformans and that CD4+ lymphocytes are critical for survival of mice infected with C. neoformans.     

A novel nervous system-induced factor inducing immune responses in the spleen

This study relates to a novel mediator signaling between the nervous system and the spleen following an immune challenge. Using enzyme-linked immunospot and cell proliferation assays, we found that supernatants of cultured splenocytes prepared from subcutaneously trypanosome-inoculated rats and mice spleens obtained immediately after inoculation and added to naive cells significantly stimulate interferon-gamma production and cell proliferation compared to phosphate-buffered saline-inoculated animals. This action was abrogated by surgical denervation of the spleen. Using the fluorescent differential display technology, the gene involved in this process was identified and further cloned and its sequence was mapped to chromosome 14 (GenBank accession number: EU552928). Protein expression revealed approximately 15 kDa molecule with biological activities similar to the cultured supernatants of splenocytes obtained directly from parasite-inoculated animals. Antibodies raised against the protein blocked the activities of both the protein and the supernatant and also recognized a band in the active supernatant with the same molecular mass as the protein. Furthermore, the protein was able to reactivate experimentally immunosuppressed cells by regaining their ability to proliferate, suggesting that such a nervous system-induced immune system-released activating agent (ISRAA) may have a potential therapeutic benefit in immunocompromised situations and in further understanding the mechanism for innate immunity commencement and action.     

Effects of Yersinia enterocolitica O:3 derivatives on B lymphocyte activation in vivo

The potential sequelae of intestinal infection with Yersinia enterocolitica include reactive arthritis, erythema nodosum, Reiter's syndrome and other autoimmune diseases. The role of the immune response in the pathogenesis of these diseases has not been fully defined, but autoimmune manifestations may be a consequence of the increase in autoantibodies as a result of polyclonal B-cell activation induced by Yersinia. We investigated the effects of Y. enterocolitica O:3 derivatives on B lymphocyte activation in vivo. Groups of five specific pathogen free (SPF) Swiss mice were inoculated with bacterial cell extract, Yersinia outermembrane proteins (Yops) or lipopolysaccharide (LPS) obtained from Y. enterocolitica O:3 and their immunoglobulin-secreting spleen cells were detected by isotype-specific protein A plaque assay. The presence of specific anti-Yersinia antibodies and autoantibodies was determined in mouse sera by ELISA. In all experiments a marked increase in the number of secretory cells of different isotypes was observed as early as the third day after inoculation. IgG and IgM anti-Yersinia antibodies were detected in the sera of all inoculated mice, and autoantibodies against myosin in the sera of those inoculated with bacterial cell extract. The sera from animals stimulated with LPS reacted with myelin, actin and laminin, while the sera from mice inoculated with Yops reacted with myelin, thyroglobulin and cardiolipin. These results suggest that SPF Swiss mice inoculated with any one of the Y. enterocolitica derivatives tested exhibited polyclonal activation of B lymphocytes as a result of stimulation by various bacterial components and not only LPS stimulation.     

Exacerbated tissue destruction in DSS-induced acute colitis of OPN-null mice is associated with downregulation of TNF-alpha expression and non-programmed cell death

Osteopontin (OPN), a pro-inflammatory mediator, is constitutively expressed in normal gut and is upregulated in inflammatory colitis. To determine the significance of OPN in inflammatory bowel disease, we studied the development of acute, experimental colitis induced by dextran sulfate sodium (DSS) in OPN-null and wild-type (WT) mice. OPN expression was markedly increased in WT diseased colons, while a higher disease activity index, including spleen enlargement, bowel shortening, and mucosal destruction, was observed in OPN-null mice. Although peripheral blood neutrophil numbers were lower in DSS-treated OPN-null mice, tissue myeloperoxidase levels, reflecting enhanced neutrophil activity, were increased in the diseased colons. In comparison, lymphocyte numbers in peripheral blood were increased earlier than in DSS-treated WT mice. Despite a significantly greater spleen enlargement, flow cytometric analysis of splenocytes from the DSS-treated OPN-null mice revealed lower numbers of differentiated macrophages and (CD4+ and CD8alpha+) lymphocytes. Whereas pro-inflammatory cytokines, including G-CSF, RANTES, MIP1alpha, and TNF-alpha, were increased < 10-fold in DSS-treated WT splenocytes, expression of these cytokines was dramatically suppressed in the DSS-treated OPN-null splenocytes as well as gut tissues. The suppressed TNF-alpha response in OPN-null mice was reflected in a marked increase in non-apoptotic cell death in diseased colons. Collectively, these studies demonstrate that OPN is required for mucosal protection in acute inflammatory colitis.     

Murine cell-mediated immune response recognizes an enterovirus group-specific antigen(s).

Splenocytes taken from mice inoculated with coxsackievirus B3 (CVB3) (Nancy) developed an in vitro proliferative response against CVB3 antigen. This response could not be detected earlier than 8 days postinoculation but could be detected up to 28 days after exposure to CB3. CVB3-sensitized splenocytes responded not only to the CVB3 antigen but to other enteroviruses as well. This response was found to be enterovirus specific in that no response was detected to a non-enteroviral picornavirus, encephalomyocarditis virus, or to an unrelated influenza virus. The generation of a splenocyte population capable of responding to an enterovirus group antigen(s) was not limited to inoculation of mice with CVB3, as similar responses were generated when mice were inoculated with CVB2. Cell subset depletions revealed that the major cell type responding to the enterovirus group antigen(s) was the CD4+ T cell. Current evidence suggests that the group antigen(s) resides in the structural proteins of the virus, since spleen cells from mice inoculated with a UV-inactivated, highly purified preparation of CVB3 virions also responded in vitro against enteroviral antigens.