Trypanosoma musculi: characterization of the T-lymphocyte dependency of immunity by selective immunomodulation of the mouse, Mus musculus

Trypanosoma musculi is a hemoflagellate parasite producing a characteristic course of infection in the mouse, terminating in elimination of the parasite from the vascular system with permanent immunity to reinfection. While this elimination is known to require functional T lymphocytes, the role of these cells has remained undefined. The current study examined the role of T-lymphocyte dependency by the selective in vivo depletion of T-lymphocyte subsets by monoclonal antibodies or immunosuppressive pharmacologic agents. In the former approach, monoclonal antibodies specific for the murine T-lymphocyte surface antigens Thy 1.2, Lyt 2, or L3T4 were administered to mice, resulting in functional depletion of either all T lymphocytes or the T-helper or T-cytotoxic subsets. Subsequent infection with T. musculi revealed that elimination of the T-helper, but not the T-cytotoxic, phenotype resulted in a prolonged and elevated parasitemia. This enhancement of parasitemia was similar to that produced with depletion of all T lymphocytes (i.e., Thy 1.2+ cells), suggesting that the T-helper cell was most important in parasite elimination. In the latter approach, pharmacologic agents displaying selective immunosuppressive activity were employed to inactivate, rather than destroy, T-lymphocyte subpopulations. In vivo inactivation of T-helper lymphocytes by the fungal metabolite cyclosporin-A enhanced the level of parasitemia in treated animals and resulted in a protracted duration of infection. In contrast, treatment with the highly substituted anthracenedione AEAD, which has been demonstrated to selectively inactivate T-cytotoxic cells, had no appreciable effect on the kinetics of infection. These results strongly suggest that the T-lymphocyte dependency of Trypanosoma musculi elimination is due predominantly to the action of the T-helper, rather than T-cytotoxic, lymphocyte.     

Functional CD8+ T cell responses in lethal Ebola virus infection

Ebola virus (EBOV) causes highly lethal hemorrhagic fever that leads to death in up to 90% of infected humans. Like many other infections, EBOV induces massive lymphocyte apoptosis, which is thought to prevent the development of a functional adaptive immune response. In a lethal mouse model of EBOV infection, we show that there is an increase in expression of the activation/maturation marker CD44 in CD4(+) and CD8(+) T cells late in infection, preceding a dramatic rebound of lymphocyte numbers in the blood. Furthermore, we observed both lymphoblasts and apoptotic lymphocytes in spleen late in infection, suggesting that there is lymphocyte activation despite substantial bystander apoptosis. To test whether these activated lymphocytes were functional, we performed adoptive transfer studies. Whole splenocytes from moribund day 7 EBOV-infected animals protected naive animals from EBOV, but not Marburgvirus, challenge. In addition, we observed EBOV-specific CD8(+) T cell IFN-gamma responses in moribund day 7 EBOV-infected mice, and adoptive transfer of CD8(+) T cells alone from day 7 mice could confer protection to EBOV-challenged naive mice. Furthermore, CD8(+) cells from day 7, but not day 0, mice proliferated after transfer to infected recipients. Therefore, despite significant lymphocyte apoptosis, a functional and specific, albeit insufficient, adaptive immune response is made in lethal EBOV infection and is protective upon transfer to naive infected recipients. These findings should cause a change in the current view of the 'impaired' immune response to EBOV challenge and may help spark new therapeutic strategies to control lethal filovirus disease.     

Site-selective homing of antigen-primed lymphocyte populations can play a crucial role in the efferent limb of cell-mediated immune responses in vivo

Lymphoid cells of the mammalian immune system exhibit special migratory properties within their in vivo environment. This fundamental characteristic is important to the protection of the organism from infection and neoplastic transformation by a process termed immunologic surveillance. The importance of lymphocyte recirculation was addressed by investigating the role of site-selective homing of lymphocytes during the efferent phase of an in vivo adoptively transferred immune response. We approached this problem by using pertussis toxin (PT), a known inhibitor of lymphoid cell migration in vivo. PT is a protein secreted by the bacterium Bordetella pertussis, which induces a selective and long-lasting inhibition of the emigration of lymphocytes from the bloodstream into solid tissue. In this study, we demonstrate that the blockade of lymphocyte extravasation potential mediates inhibition of certain cell-mediated immune responses in vivo. We investigated the effect of PT on the extravasation of antigen-primed lymphocytes into solid tissue. The results show that the loss of lymphocyte homing potential after in vitro treatment of the primed cells with PT is accompanied by an inhibition of antigen-specific contact hypersensitivity after adoptive transfer. This result suggests that the process of lymphocyte extravasation into nonlymphoid tissue sites such as the skin shares fundamental similarities to the selective localization of circulating lymphocytes to lymph nodes. Furthermore, the inhibition of contact hypersensitivity observed after the i.v. adoptive transfer of PT-treated antigen-primed cells could be circumvented by transferring the PT-treated cells directly into a tissue site with the relevant antigen. In contrast, no inhibition in the number of antibody-forming cells present within the spleen was observed after an adoptive transfer of PT-treated sheep red blood cell-primed lymphocytes, a result that is in agreement with radiotracer data. Thus, the inhibitory effect of PT on the adoptive transfer of contact hypersensitivity was established to be a direct result of the PT-mediated alteration of cellular migration, because PT inhibits the entrance of lymphocytes into specific tissue sites without inhibiting other cellular functions. This conclusion is additionally supported by experiments that showed that lymphocytes that have been pretreated with PT exhibit normal in vitro responses, as assayed by mitogenesis in response to concanavalin A and by the differentiation and function of alloantigen stimulated or hapten-specific cytotoxic T lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adoptive transfer of NK 1.1+ lymphocytes in immune-mediated colitis: a pro-inflammatory or a tolerizing subgroup of cells?

T lymphocytes expressing NK1.1 marker (NKT) have been suggested to play crucial roles in immune modulation. AIM: To determine the role of NK1.1+ cells in induction and maintenance of pro-inflammatory and/or tolerizing responses. METHODS: Colitis was induced in C57/B6 donor mice by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Donor mice received five oral doses of colonic proteins extracted from TNBS-colitis colonic wall. Depletion of NK1.1+ lymphocytes was performed before lymphocyte harvesting. Splenocytes were harvested and separated into T-cell subpopulations, and transplanted into recipient mice before intracolonic instillation of TNBS. Standard clinical, macroscopic, and microscopic scores, and intracellular staining, flow cytometry, and cytotoxicity assays were performed. RESULTS: The adoptive transfer of CD4+ and NK1.1+ cells harvested from tolerized mice markedly ameliorated the colitis in recipient mice. In contrast, the adoptive transfer of CD8+ and double negative lymphocytes failed to transfer the tolerance. Recipients of splenocytes from tolerized mice exhibited an increase in CD4+ IL4+/CD4+ IFNgamma+ ratio. In contrast, recipients of splenocytes from NK1.1-depleted-tolerized mice exhibited severe colitis with a significant decrease of the CD4+ IL4+/CD4+ IFNgamma+ ratio. However adoptive transfer of splenocytes from non-tolerized NKT-depleted mice led to an alleviation of colitis with a relative increase of the CD4+ IL4+/CD4+ IFNgamma+ ratio. CONCLUSIONS: NK1.1+ lymphocytes play a critical role in immune regulation. They may be accountable for an alteration of the inflammatory response and the CD4+ IL4+/CD4+ IFNgamma ratio immune-mediated colitis and in peripheral tolerance induction.     

Effective clearance of a persistent viral infection requires cooperation between virus-specific Lyt2+ T cells and nonspecific bone marrow-derived cells

The lifelong chronic lymphocytic choriomeningitis virus (LCMV) infection established in neonatally or congenitally infected mice can be eliminated by adoptive transfer of lymphoid cells from LCMV-immune mice. In this study, we have identified the effector cells mediating the clearance of persistent and disseminated LCMV infection. Using mice that are recombinant in the H-2 region and by selective depletion of lymphocyte subpopulations, we show that viral clearance was mediated by LCMV-specific Lyt2+ L3T4- T cells that are restricted to the class I genes of the major histocompatibility complex. In addition, our results show a requirement for host-derived bone marrow cells for the effective elimination of virus from the liver. These studies emphasize the importance of virus-specific T cells and an intact bone marrow function in viral clearance.     

Selective growth, in vitro and in vivo, of individual T cell clones from tumor-infiltrating lymphocytes obtained from patients with melanoma

In recent clinical trials in patients with metastatic melanoma, adoptive transfer of tumor-reactive lymphocytes mediated the regression of metastatic tumor deposits. To better understand the role of individual T cell clones in mediating tumor regression, a 5' RACE technique was used to determine the distribution of TCR beta-chain V region sequences expressed in the transferred cells as well as in tumor samples and circulating lymphocytes from melanoma patients following adoptive cell transfer. We found that dominant T cell clones were present in the in vitro-expanded and transferred tumor-infiltrating lymphocyte samples and certain T cell clones including the dominant T cell clones persisted at relatively high levels in the peripheral blood of the patients that demonstrated clinical responses to adoptive immunotherapy. However, these dominant clones were either undetected or present at a very low level in the resected tumor samples used for tumor-infiltrating lymphocyte generation. These data demonstrated that there was selective growth and survival, both in vitro and in vivo, of individual T cell clones from a relatively small number of T cells in the original tumor samples. These results suggest that the persistent T cell clones played an active role in mediating tumor regression and that 5' RACE analysis may provide an important tool for the analysis of the role of individual T cell clones in mediating tumor regression. A similar analysis may also be useful for monitoring autoimmune responses.     

Gamma delta T cell-deficient mice have a down-regulated CD8+ T cell immune response against Encephalitozoon cuniculi infection

Gamma(delta) T cells have been reported to play an essential effector role during the early immune response against a wide variety of infectious agents. Recent studies have suggested that the gamma(delta) T cell subtype may also be important for the induction of adaptive immune response against certain microbial pathogens. In the present study, an early increase of gamma(delta) T cells during murine infection with Encephalitozoon cuniculi, an intracellular parasite, was observed. The role of gamma(delta) T cells against E. cuniculi infection was further evaluated by using gene-knockout mice. Mice lacking gamma(delta) T cells were susceptible to E. cuniculi infection at high challenge doses. The reduced resistance of delta(-/-) mice was attributed to a down-regulated CD8+ immune response. Compared with parental wild-type animals, suboptimal Ag-specific CD8+ T cell immunity against E. cuniculi infection was noted in delta(-/-) mice. The splenocytes from infected knockout mice exhibited a lower frequency of Ag-specific CD8+ T cells. Moreover, adoptive transfer of immune TCR(alpha)beta+ CD8+ cells from the delta(-/-) mice failed to protect naive CD8(-/-) mice against a lethal E. cuniculi challenge. Our studies suggest that gamma(delta) T cells, due to their ability to produce cytokines, are important for the optimal priming of CD8+ T cell immunity against E. cuniculi infection. This is the first evidence of a parasitic infection in which down-regulation of CD8+ T cell immune response in the absence of gamma(delta) T cells has been demonstrated.     

The age-related resistance of rats to Plasmodium berghei infection is associated with differential cellular and humoral immune responses

In this study, we investigated how the age of rats would affect the course of infection of and the immune response to Plasmodium berghei. Both young (4-week-old) and adult rats (8-week-old) can be infected with P. berghei ANKA strain, with significantly higher levels of infected red blood cells in young rats. While 100% of young rats succumbed to infection, adult rats were able to clear blood parasites and no mortality was observed. Analysis of cellular distribution and circulating cytokines demonstrated the persistence of CD4+/CD25+ T cells and high expression of circulating interleukin-10 (IL-10) during the progression of infection in young-susceptible rats, whereas high levels of CD8+ T cells and natural killer T cells are detected in adult-resistant rats. Analysis of antibody isotypes showed that adult rats produced significantly higher levels of interferon-gamma (IFN-gamma)-dependent IgG2c antibodies than young rats during infection. Further evaluation of the role of IL-10, IFN-gamma and of immune cells showed that only the adoptive transfer of spleen cells from adult-resistant rats was able to convert susceptibility of young-susceptible rats to a resistant phenotype. These observations suggest that cell-mediated mechanisms are crucial for the control of a primary infection with P. berghei in young rats.     

Efficacious control of cytomegalovirus infection after long-term depletion of CD8+ T lymphocytes.

Although the relative contribution of different immune effector functions to clearing tissues of cytomegalovirus is controversial, the contribution of CD8+ T lymphocytes has generally been accepted as essential. In this report, we show that under certain conditions the CD8+ T-lymphocyte subset can be dispensable for clearance of cytomegalovirus. Mice depleted of the CD8+ T-lymphocyte subset eliminated infectious virus with a clearance kinetics similar to that of normal mice. Adoptive transfer studies revealed that the limitation of virus spread required the cooperation between the CD4+ subset and other cells. Comparison between protective functions generated in fully immunocompetent and in CD8- mice demonstrated that elimination of the CD8+ subset before infection altered the quality of the antiviral immune response. The compensatory protective activity gained by CD4+ cells in CD8- mice was absent in normal mice recovering from virus infection.     

Human CD4+ T cells mediate rejection of porcine xenografts

It has previously been demonstrated that xenograft rejection in rodents is dependent on CD4+ T cells. However, because of the lack of an appropriate in vivo model, little is known about the cellular basis of human T cell-mediated rejection of xenografts. In this study, we have evaluated the ability of human T cells to mediate rejection of porcine skin grafts in a novel in vivo experimental system using immunodeficient mice as recipients. Recombinase-activating gene-1-deficient mice (R-) lacking mature B and T cells were grafted with porcine skin and received human lymphocytes stimulated in vitro with irradiated porcine PBMC. Skin grafts on mice given either unseparated, activated human lymphocytes, or NK cell-depleted lymphocyte populations were rejected within 18 days after adoptive cell transfer. In contrast, skin grafts on mice given T cell-depleted human lymphocytes or saline showed no gross or histologic evidence of rejection up to 100 days after adoptive transfer. Purified CD4+ T cells were also able to mediate rejection of porcine skin grafts. These data suggest that human CD4+ T cells are sufficient to induce rejection of porcine xenografts. Thus, strategies directed toward CD4+ T cells may effectively prevent cellular rejection of porcine xenografts in humans.     

Transfer of immunity to Babesia microti of human origin using T lymphocytes in mice

Lymph node and spleen cells from mice infected with Babesia microti of human origin developed the ability to transfer adoptive immunity to naive mice within 25 days after infection. This protective activity was greater in cells obtained at 32 days than in cells obtained at 25 days postinfection and remained stable up to 52 days postinfection. Recipients of lymph node cells and spleen cells displayed similar peak parasitemias although 2 days after peak parasitemia, immune spleen cell recipients had significantly lower parasitemias than immune lymph node cell recipients. Strong protective activity was demonstrated when cells were transferred 1 day postinfection, while equal numbers of cells, transferred 3 days postinfection did not confer significant protection over nonimmune cells. There was also a suggestion that the number of immune spleen cells necessary for significant protection was directly related to the number of parasites inoculated. The subpopulation of lymphocytes responsible for the transfer of adoptive immunity to B. microti of human origin was then studied in BALB/c mice depleted of T lymphocytes by thymectomy and lethal irradiation. One day after infection with B. microti, T-cell-depleted mice were given complement-treated immune spleen cells, anti-θ serum-treated immune spleen cells, nonimmune spleen cells, or no cells. Similar experiments were performed comparing the effects of anti-immunoglobulin serum-treated and unfractionated immune spleen cells on B. microti parasitemia. Treatment with anti-θ serum abrogated the protective activity of immune spleen cells while anti-immunoglobulin serum treatment had no effect. These results suggest that immunologic memory of B. microti in BALB/c mice is modulated by T rather than B lymphocytes.     

Recovery from chronic rotavirus infection in mice with severe combined immunodeficiency: virus clearance mediated by adoptive transfer of immune CD8+ T lymphocytes.

Severe combined immunodeficient (SCID) mice lack both functional T and B cells. These mice develop chronic rotavirus infection following an oral inoculation with the epizootic diarrhea of infant mice (EDIM) rotavirus. Reconstitution of rotavirus-infected SCID mice with T lymphocytes from immunocompetent mice allows an evaluation of a role of T-cell-mediated immunity in clearing chronic rotavirus infection. Complete rotavirus clearance was demonstrated in C.B-17/scid mice 7 to 9 days after the transfer of immune CD8+ splenic T lymphocytes from histocompatible BALB/c mice previously immunized intraperitoneally with the EDIM-w strain of murine rotavirus. The virus clearance mediated by T-cell transfer was restricted to H-2d-bearing T cells and occurred in the absence of rotavirus-specific antibody as determined by enzyme-linked immunosorbent assay, neutralization, immunohistochemistry, and radioimmunoprecipitation. Temporary clearance of rotavirus was observed after the transfer of immune CD8+ T cells isolated from the intestinal mucosa (intraepithelial lymphocytes [IELs]) or the spleens of BALB/c mice previously infected with EDIM by the oral route. Chronic virus shedding was transiently eliminated 7 to 11 days after spleen cell transfer and 11 to 12 days after IEL transfer. However, recurrence of rotavirus infection was detected 1 to 8 days later in all but one SCID recipient receiving cells from orally immunized donors. The viral clearance was mediated by IELs that were both Thy1+ and CD8+. These data demonstrated that the clearance of chronic rotavirus infection in SCID mice can be mediated by immune CD8+ T lymphocytes and that this clearance can occur in the absence of virus-specific antibodies.     

Molecules that inhibit T-cell functions: cytochemical localization and shuttling

Adaptive immune responses to antigens are mediated by specific receptors expressed on B cells (BCR's) and T cells (TCR's). Effector cells and memory cells are produced following a proliferative wave that accounts for clonal expansion. If not down-regulated, clonal expansion might lead to uncontrolled lymphoproliferation that would be harmful for the organism. Several mechanisms that account for the down-sizing of activated lymphocyte clones are briefly reviewed here. We next consider in detail one such mechanism that deals with the functional characterization and the immunocytochemical localization of two T-cell inhibitory molecules, namely the Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) and the HP-F1 antigen, both present in all T lymphocytes. CTLA-4 and HP-F1 inhibit CD4+ T-helper cell proliferation and the lytic ability of CD8+ T-cytotoxic cells in non-specific and in antigen-specific cytolytic assays. Interestingly, a clonal distribution exists as for the ability of CTLA-4 and HP-F1 to inhibit T-cell functions. In resting and activated T cells, both molecules are largely confined in the endosomal compartment, as shown by immunofluorescence analyses. However, upon interaction of T cells with Antigen-Presenting Cells (APC's) or with target cells that must be killed, CTLA-4 molecules are transported to the plasma membrane, at the site of cell-to-cell contact where, following interaction with ligands, they trigger inhibitory signals.     

B lymphocytes are required for the generation of T cells that mediate healing of cutaneous leishmaniasis

The role that B lymphocytes and/or antibodies play in the healing of Leishmania major infections in genetically resistant C3H/HeN mice was investigated by monitoring the course of infection in animals that had been B cell depleted by treatment from birth with anti-IgM sera (mu-suppressed). L. major infection of mu-suppressed C3H/HeN mice produced lesions that were significantly larger than those induced in control animals, and failed to heal. Moreover, vaccinated mu-suppressed mice also developed chronic nonhealing infections, although their lesions were initially smaller than those developed by nonvaccinated mu-suppressed controls. The enhanced susceptibility of mu-suppressed mice could be completely overcome by adoptive transfer of T lymphocytes from mice that had spontaneously healed their lesions, and to a lesser extent by T lymphocytes from normal animals. Anti-leishmanial antibody responses were completely absent in mu-suppressed mice, regardless of whether they were lymphocyte reconstituted, whereas delayed type hypersensitivity (DTH) to leishmanial antigens was present in normal and mu-suppressed animals. The ability of immune T cells to protect mu-suppressed mice without restoring humoral responsiveness clearly indicates that antibodies are not necessary for healing leishmanial infections. Instead, the observed effect of mu-suppression argues that B lymphocytes are required for the generation of an effector T cell population, apparently unrelated to DTH, which mediates the healing of cutaneous lesions. These results thus provide the first evidence for the B cell and/or Ig dependency of a T cell population that is critical for the development of immunity against a microbial agent.     

Trypanosoma musculi: population dynamics of erythrocytes and leukocytes during the course of murine infections

Cells of the hemocytic and lymphoreticular series located in the blood, bone marrow, spleen, and peritoneal space have been analyzed throughout the course of Trypanosoma musculi infections of intact and splenectomized C3H female mice. Following an early (within 2 days after trypanosome inoculation intraperitoneally) shift of leukocytes from the blood to the peritoneal space, there occurred a more gradual, prolonged infusion of leukocytes into the peritoneal space, the primary site of infection, that continued until the infection was terminated. There was intense cytogeneractive activity in the spleen that included erythrocytes, lymphocytes, myelocytes, and megakaryocytes. The marrow became primarily a site of monocytopoiesis and, to some extent, of lymphopoiesis. During the first 8 days (approximately) of infection, there was a decline in mature erythrocytes in the blood (the well-known anemia) and development of a profound thrombocytopenia. In splenectomized mice, the depletion of these elements continued unabated until the mice died; the marrow of infected, splenectomized mice failed to provide these elements, as was also the case in intact mice. In the peritoneal space, the intense battle between leukocytes and trypanosomes was reflected in a gradual, impressive rise in the number of dead and fatigued cells and, late in infection, in the development of ascites. Both of these abnormal conditions disappeared shortly after cure of the infection. We conclude that infections of mice with T. musculi result in dedication of the entire lymphoreticular system to the generation of cells that are exported to the peritoneal space to combat the major infection the occurs in that locale. This is consistent with the evidence that the belated immune elimination of T. musculi is a cell-mediated (probably antibody-dependent) process. The disruption of the normal histoarchitecture, the shift in the normal proportions of cells and in cells of different degrees of maturity, and probably, a block imposed on precursor cell maturation, account to a large extent for the well-known failure of immune responses commonly associated with trypanosome infections.     

T cytotoxic-1 CD8+ T cells are effector cells against pneumocystis in mice

Host defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes. A hallmark of HIV infection is Pneumocystis carinii (PC) pneumonia. Recently, CD8+ T cells, which are recruited to the lung in large numbers in response to PC infection, have been associated with some level of host defense as well as contributing to lung injury in BALB/c mice. In this study, we show that CD8+ T cells that have a T cytotoxic-1 response to PC in BALB/c mice, as determined by secretion of IFN-gamma, have in vitro killing activity against PC and effect clearance of the organism in adoptive transfer studies. Moreover, non-T cytotoxic-1 CD8+ T cells lacked in vitro effector activity and contributed to lung injury upon adoptive transfer. This dichotomous response in CD8+ T cell response may in part explain the clinical heterogeneity in the severity of PC pneumonia.     

Immunization with baculovirus-expressed recombinant rotavirus proteins VP1, VP4, VP6, and VP7 induces CD8+ T lymphocytes that mediate clearance of chronic rotavirus infection in SCID mice.

Clearance of chronic murine rotavirus infection in SCID mice can be demonstrated by adoptive transfer of immune CD8+ T lymphocytes from histocompatible donor mice immunized with a murine homotypic rotavirus (T. Dharakul, L. Rott, and H.B. Greenberg, J. Virol 64:4375-4382, 1990). The present study focuses on the protein specificity and heterotypic nature of cell-mediated clearance of chronic murine rotavirus infection in SCID mice. Heterotypic cell-mediated clearance was demonstrated in SCID mice infected with EDIM (murine) rotavirus after adoptive transfer of CD8+ T lymphocytes from BALB/c mice that were immunized with a variety of heterologous (nonmurine) rotaviruses including Wa (human, serotype 1), SA11 and RRV (simian, serotype 3), and NCDV and RF (bovine, serotype 6). This finding indicates the serotypic independence of T-cell-mediated rotavirus clearance. To further identify the rotavirus proteins that are capable of generating CD8+ T cells that mediate virus clearance, donor mice were immunized with SF-9 cells infected with a baculovirus recombinant expressing one of the following rotavirus proteins: VP1, VP2, NS53 (from RF), VP4, VP7, NS35 (from RRV), VP6, and NS28 (from SA11). SCID mice stopped shedding rotavirus after receiving CD8+ T cells from mice immunized with VP1, VP4, VP6, and VP7 but not with VP2, NS53, NS35, NS28, or wild-type baculovirus. These results suggest that heterotypic cell-mediated clearance of rotavirus in SCID mice is mediated by three of the major rotavirus structural proteins and by a putative polymerase protein.     

In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection

Injection of CBA mice with Brucella abortus strain 19 leads to chronic infection during which both cell-mediated immunity (delayed hypersensitivity and macrophage activation) and antibody production occur. Protection was efficiently transferred to naive mice using spleen cells from mice infected 5 or 12 weeks earlier. Selective lysis in vitro of these cells by antibody to cell surface antigens showed that Thy-1⁺ Ly-1⁺2⁺ T lymphocytes were required for transfer. Treatment with anti-Ia serum neither suppressed nor enhanced adoptive transfer. Thus Ia⁺ B lymphocytes were not required, and Ia⁺ suppressor T cells were not active in the response. Three injections per week of anti-Ly-1 monoclonal antibody beginning 5 days before infection led to a 10-fold increase in bacterial numbers 25 days after infection when acquired immunity was well established in untreated mice. The delayed hypersensitivity response was unaffected. In addition cells from these in vivo treated mice were unable to transfer resistance. Beginning the treatment on the day of infection abolished the IgG antibody response without affecting bacterial numbers. The studies emphasize the unique role of Ly-1⁺2⁺ T cells in immunity to Brucella and indicate the usefulness of these techniques in dissecting out those components of the immune response which contribute to recovery from infection.     

Cellular subsets involved in cell-mediated immunity to murine Plasmodium yoelii 17X malaria

Cell mediated immunity to nonlethal Plasmodium yoelli 17X (PY17X-NL) was examined in the CBA/CaJ mouse by adoptive transfer of sensitized T lymphocyte subsets. In intact mice, PY17X-NL causes a self-limiting infection with parasitemia levels ranging from 10 to 25% of total red blood cells. Upon recovery, mice are refractory to subsequent challenge with the homologous parasite. In T cell-depleted mice, PY17X-NL infections are extremely virulent and result in death of the host after parasitemia levels reach 50% or higher. The transfer of either Lyt-1 T cells or Lyt-2 T cells from immune animals into normal, naive animals produced accelerated recovery to subsequent infection. However, this adoptive transfer of immunity by either subset was dependent upon the presence of an I-J+, Lyt-null cell in the immune population. T cell deprivation precluded the ability of animals to control blood-stage infections. When T cell-depleted mice were reconstituted with naive, Ig-negative (T cell-enriched) spleen cells, parasitemia levels were controlled and the parasites were eliminated. When T cell-deprived animals were reconstituted with naive Lyt-1+2-, Ig-negative spleen cells, they experienced twofold higher parasitemias of longer duration than mice receiving unfractionated cells. Two of six of these Lyt-1 mice died of fulminant infections, suggesting that the presence of naive Lyt-2 cells enhances the degree of protection. Immune Lyt-2 T cells were highly protective in T cell-depleted animals. Protection by sensitized Lyt-1 T cells correlated with the induction of a monocytosis. On the other hand, protection by Lyt-2T cells occurred in the absence of monocytosis. The possibility that the immunity induced by each T cell subset is mediated by a different effector mechanism is discussed.     

Cellular basis of immunologic interactions in adoptive T cell therapy of established metastases from a syngeneic murine sarcoma

The adoptive transfer of specifically sensitized T lymphocytes can effectively mediate the regression of established local and metastatic tumors. Previous experiments using the weakly immunogenic MCA 105 sarcoma indicated that cellular interactions between transferred L3T4+ helper and Lyt-2+ cytotoxic immune T cells were necessary for mediating tumor regression. In this study, the kinetics of T-T cell interactions were analyzed by in vivo depletion of T cell subsets with mAb. The anti-tumor efficacy of transferred immune cells was abrogated by in vivo administration of either L3T4 or Lyt-2 mAb on the day of cellular therapy. However, if mAb were given 3 days after the transfer of immune cells, depletion of Lyt-2+ but not L3T4+ cells abrogated anti-tumor efficacy. T cell depletion on day 6 after transfer of immune cells had no adverse effect on tumor regression, indicating the period required for T cell reactivity in vivo. Furthermore, depletion of Ia+ cells by in vivo mAb treatment abrogated the anti-tumor efficacy of immune cells. It is thus hypothesized that there are two distinct but sequential phases of in vivo T cell interactions leading to the regression of established tumors after adoptive immunotherapy. An initial "helper/inducer" phase apparently requires the interaction of L3T4+ immune cells and the tumor Ag involving Ia+ cells. The inducement of L3T4+ cell activation is to provide helper function via the secretion of IL-2. The second phase designated as an "effector phase" involves differentiation of immune Lyt-2+ cells under the influence of IL-2 secreted during the helper/inducer phase for generation of mature Lyt-2+ effector cells. To further support the hypothesis of a two-phase process we have examined the phenotype and kinetics of tumor regression mediated by effector cells generated by secondary in vitro sensitization (IVS). Although the IVS cells were generated from fresh MCA 105 immune spleen cells, their anti-tumor efficacy was mediated solely by Lyt-2+ lymphocytes. Kinetic studies revealed that the in vivo requirement of IVS Lyt-2+ effector cells to mediate tumor regression was less than 3 days, and the anti-tumor reactivity of these cells was not affected by in vivo depletion of Ia+ cells. Thus, the IVS reaction is likely representative of the in vivo counterpart of the helper/inducer phase leading to the generation of mature Lyt-2+ immune effector cells. Tumor regression after transfer of Lyt-2+ cells generated in IVS therefore required a relatively shorter period of time than that required after the transfer of fresh noncultured MCA 105 immune spleen cells.