Antigen processing of vesicular stomatitis virus in situ. Interdigitating dendritic cells present viral antigens independent of marginal dendritic cells but fail to prime CD4(+) and CD8(+) T cells.

Acute macrophage (M phi) depletion, using a liposome-mediated 'suicide technique', markedly suppressed priming of splenic CD4(+) and CD8(+) T-cell responses to vesicular stomatitis virus (VSV). However, phagocytic marginal dendritic cells (MDC), but not interdigitating dendritic cells (IDC), are now known to be also depleted by this technique. To clarify the role splenic dendritic cell (DC) subsets and M phi play in priming for a virus-specific T-cell-mediated immune response, DC and M phi were purified from VSV-infected mice and assayed for the presence of epitopes recognized by VSV helper T (Th) cells and cytotoxic T lymphocytes (CTL). Antigen pulse experiments performed in situ demonstrated that VSV Th cell and CTL epitopes became transiently associated only with DC, but not M phi or B cells, indicating that DC represent the critical antigen-presenting cell (APC) population in vivo for this virus. The failure of MDC/M phi-deficient mice to become primed was not due to the complete elimination of antigen-presenting DC because VSV peptide/class I and II complexes were detected on IDC following lipsome-mediated elimination of phagocytic cells. However, the VSV-induced chemokine response was dramatically suppressed in these mice. Thus, despite the expression of VSV peptide/class I and II complexes, IDC are not sufficient to prime VSV Th cells in the absence of MDC and/or splenic M phi.

Flt3-ligand induces transient tumor regression in an ectopic treatment model of major histocompatibility complex-negative prostate cancer.

We assessed the in vivo efficacy of Flt3-ligand (Flt3-L) treatment in C57BL/6 mice bearing a well-established MHC class I-negative prostate carcinoma TRAMP-C1. Flt3-L immunotherapy was initiated approximately 30 days after tumor inoculation, a time when > or =80% of the mice had palpable TRAMP-C1 tumors. Treatment with Flt3-L at 10 microg/day for 21 consecutive days suppressed TRAMP-C1 tumor growth and induced tumor stabilization (P = 0.0337). Enhanced tumor regression was demonstrated at a higher dose of 30 microg/day (P < 0.0001). Tumors excised from mice treated with Flt3-L were smaller than carrier-treated controls and contained a more pronounced mixed inflammatory cell infiltrate primarily composed of mphi. In regressor nice, tumors reappeared at the site of injection when Flt3-L therapy was terminated. When the experiment was repeated with MHC class I-positive TRAMP-C1 cells, tumor stabilization and/or regression was again observed after treatment (P < 0.0001); however, once again, tumors reappeared after the termination of therapy despite an extended treatment schedule (35 days). MHC class I-negative variants were present in tumors isolated from carrier- and Flt3-L-treated mice, and this phenotype could be reversed by IFN-gamma treatment in vitro. Thus, Flt3-L treatment of mice with preexisting transplantable prostate tumors results in tumor regression that is dose-dependent and accompanied by a pronounced mixed-cell inflammatory tumor infiltrate. However, disease relapse was invariably observed after the termination of therapy, which suggests that Flt3-L treatment of advanced MHC- prostate cancers will require adjuvant modalities to achieve a durable response.

Three subpopulations of fast axonally transported retinal ganglion cell proteins are differentially trafficked in the rat optic pathway.

Post-Golgi trafficking of the major fast axonally transported (FT) proteins was investigated in the rat optic pathway. Following intra-ocular injection of 35S-methionine, radiolabeled FT proteins in the optic tract (OT) and superior colliculus (SC) were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. Twenty FT proteins, including a known plasma membrane protein (SNAP-25) and synaptic vesicle protein (synaptobrevin-2), displayed consistent 2D-PAGE migration behavior and were chosen for densitometric quantitative analysis. Results showed that at least three subpopulations of the 20 FT proteins could be differentiated based on their trafficking behavior to axons (OT) vs. terminals (SC). To assess whether Golgi-independent processes (e.g., delayed somal release and/or retrograde transport) could account for the differential compartmentation behavior between the three FT classes, we assessed whether radiolabeled FT proteins became redistributed in the optic pathway following a nerve transection blockade. The results showed that radiolabelled FT proteins did not show a quantitative change in their axon vs. terminal compartmentation in response to disconnection from cell bodies or targets. Thus, the three classes of fast axonally transported proteins were likely trafficked to distinct destinations in the optic pathway by Golgi sorting mechanisms.

Replication of murine cytomegalovirus in differentiated macrophages as a determinant of viral pathogenesis.

Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as "filters" in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection.

Differential effects of axotomy on the in vivo synthesis of the stress-inducible and constitutive 70-kDa heat-shock proteins in rat dorsal root ganglia.

The purpose of this study was to the test the hypothesis that heat-shock protein expression is upregulated (or induced) in dorsal root ganglia (DRG) following axotomy. To test this hypothesis, DRG or sciatic nerve (SN) proteins were pulse-labelled in vivo with [35S]methionine and the metabolic synthesis of two major 70-kDa heat-shock proteins, the constitutive species (hsc70) and stress-inducible species (hsp68), were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. Results showed that DRG hsp68 expression was absent (or barely detectable) under normal (sham-axotomy) conditions. However, following long-range axotomy (35 mm from DRG), there was a delayed (> 12 h post-axotomy) and transient upregulation of DRG hsp68 metabolic synthesis. Control studies demonstrated that, although DRG hsp68 was upregulated, hsp68 was not induced in SN regions proximal to the crush site. In contrast to DRG hsp68 expression, there was abundant DRG hsc70 synthesis under normal conditions that did not significantly change following axotomy. These results suggest that a specific stress protein response is induced in DRG following axotomy.

T cell priming against vesicular stomatitis virus analyzed in situ: red pulp macrophages, but neither marginal metallophilic nor marginal zone macrophages, are required for priming CD4+ and CD8+ T cells.

Since extensive degradation may be required to present complex Ags, we addressed whether macrophages (M phi) might function as APC for anti-viral cell-mediated immune responses. To study this question, murine splenic M phi were depleted by i.p. administration of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP-liposomes or clodronate-liposomes) before priming mice with vesicular stomatitis virus (VSV). Cl2MDP-liposome treatment resulted in the rapid (1-day) depletion of splenic M phi that was associated with a suppression of the ability of M phi-deficient mice to generate secondary anti-VSV CTL and Th cell proliferative responses in vitro. Control studies demonstrated that splenic dendritic cells were not adversely affected by treatment with Cl2MDP-liposomes. To assess the contribution of splenic M phi subpopulations to T cell priming against this virus, priming was delayed following treatment with Cl2MDP-liposomes until specific M phi subsets had repopulated the spleen. This analysis revealed that repopulation by red pulp M phi, but not with other splenic M phi subsets, was associated with the ability to mount normal secondary CTL and Th cell responses against VSV. Depletion of splenic, but not resident, peritoneal M phi by i.v. injection of Cl2MDP-liposomes did not rescue T cell priming in VSV-infected mice. Thus, only red pulp M phi, and not other splenic or peritoneal M phi populations, are necessary for T cell priming to VSV, a biochemically complex Ag.

Priming antiviral cytotoxic T lymphocytes: requirement for CD4+ cells is dependent on the antigen presenting cell in vivo.

We have analyzed cytotoxic thymus-derived lymphocyte (CTL) responses to vesicular stomatitis virus (VSV) to determine whether VSV precursor CTL (pCTL) can be primed in vivo in the absence of CD4+ cells. Our studies demonstrated that secondary anti-VSV CTL responses in vitro were markedly reduced by CD4-depletion prior to priming in vivo with VSV. Limiting dilution analysis indicated that the vast majority (> 90%) of VSV pCTL failed to become primed when exposed to VSV in the absence of CD4+ cells. A second minor population (5-10%) of pCTL was identified that was reproducibly primed in CD4-deficient mice. In contrast to CD4-depleted mice infected with free, infectious virus, CD4-deficient mice primed with VSV-infected, activated B cells mounted normal secondary anti-VSV CTL responses in vitro. Precursor estimates indicated that virtually all VSV pCTL became primed using this cellular immunogen. CD4-independent priming could not be achieved using VSV-infected, activated T cells, another permissive cell type for VSV replication. Thus, most VSV pCTL require inductive signals from classical CD4+ helper T cells in order to become primed in vivo and this requirement may be regulated in vivo by the antigen presenting cell.

Heat stress induces hsc70/nuclear topoisomerase I complex formation in vivo: evidence for hsc70-mediated, ATP-independent reactivation in vitro.

We previously demonstrated that in murine T cells thermotolerance correlated with heat shock protein 70 (hsp70) synthesis and protection of nuclear type I topoisomerase (topo I). Topo I activity returned to normal levels following heat stress even in cells not rendered thermotolerant by a prior heat shock. Recovery of topo I activity was not dependent on de novo protein synthesis, suggesting that the cell possesses a pathway(s) for refolding this nuclear protein. In this report we demonstrate that topo I and hsc70, the constitutively produced member of the hsp70 family, associated in vivo during heat stress. That this association may play a physiologically important role in protecting topo I activity from heat stress was suggested by the observation that hsc70 protected topo I from heat inactivation in vitro. hsc70 but not actin also reactivated previously heat-denatured topo I in a dose-dependent fashion. However, refolding of heat-denatured topo I by purified hsc70 was inefficient relative to a hsc70-containing cell lysate. Protection from heat inactivation as well as reactivation by hsc70 did not require exogenous ATP. Similarly, reactivation by the cell lysate was not inhibited by ADP or a nonhydrolyzable analogue of ATP. Thus, our studies suggest that nuclear topo I complexes with hsc70 during heat stress, which may explain, at least in part, why hsp70 proteins accumulate in the nucleus, particularly the nucleolus. This interaction may limit heat-induced protein damage and/or accelerate restoration of protein function in an ATP-independent reaction.

Induction of thermotolerance in T cells protects nuclear DNA topoisomerase I from heat stress.

In this study, we have demonstrated that topoisomerase I DNA relaxing activity is protected against a severe heat shock in T cells made thermotolerant by a prior modest heat treatment. However, following a severe heat-shock challenge and incubation at 37 degrees C, topoisomerase activity in the control population eventually returned to levels similar to those detected in thermotolerant cells. This recovery of topoisomerase activity appears to result from the renaturation of heat-inactivated enzyme rather than from synthesis of new protein because the rate of recovery of catalytic activity was not inhibited by the presence of the protein synthesis inhibitor, cycloheximide.

T helper cells in cytotoxic T lymphocyte development: analysis of the cellular basis for deficient T helper cell function in the L3T4-independent T helper cell pathway.

In this present study, lymphokine (IL-2/IL-4) production in VSV-induced Th cell (L3T4+Lyt-2- VSV-immune T cells) and memory CTL populations (L3T4-Lyt-2+ VSV-immune T cells) has been assessed in order to gain some understanding as to why the Lyt-2+ subset (L3T4-independent Th cell pathway) fails to provide Th cell function for anti-VSV CTL responses. Our studies demonstrated that following specific antigen (VSV, H-2 antigen) or mitogen stimulation, lymphokine activity was detected in the supernatants obtained from VSV-induced Th but not VSV memory CTL populations. The presence of blocking concentrations of PC61, a monoclonal antibody (mAb) to the IL-2 receptor (IL-2R), revealed augmented lymphokine activity only in the VSV-induced Th cell supernatant. VSV-induced Th cells secreted both IL-2 and IL-4 following stimulation with VSV. Two lines of evidence supported the view that both these lymphokines were important for an anti-VSV CTL response: (1) mAb to either IL-2 or IL-4 inhibited CTL maturation and (2) the combination of exogenous IL-2 and IL-4 reconstituted a class I-restricted. VSV-specific CTL response in Th cell-depleted T cell cultures. The failure to detect lymphokine production in bulk cultures of the VSV memory CTL population was consistent with limiting dilution (LD) analysis of lymphokine-producing cells in the spleen of VSV-immune mice. Thus, approximately 1/15,000 Lyt-2-depleted, VSV-immune T cells were positive for lymphokine production following VSV stimulation, whereas less than 1/1,000,000 L3T4-depleted, VSV-immune T cells were scored as lymphokine-secreting cells following stimulation with this same virus. Similarly, precursor estimates for lymphokine-producing cells against allogeneic class I antigens demonstrated that the majority of lymphokine-producing cells also resided in the L3T4+ subset. Lymphokine-secreting Lyt-2+ cells were detected at low but not high cell densities suggesting that Lyt-2+ cells may secrete another lymphokine(s) that inhibits IL-2/IL-4 production. Thus, these studies demonstrate an obligatory requirement for the L3T4-dependent Th cell pathway for optimal CTL responses derived from either CTLp or memory CTLs.

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress.

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.

T lymphocyte stress response. I. Induction of heat shock protein synthesis at febrile temperatures is correlated with enhanced resistance to hyperthermic stress but not to heavy metal toxicity or dexamethasone-induced immunosuppression.

We have investigated the effect of febrile temperatures (less than or equal to 41 degrees C) on T cell heat shock protein (hsp) synthesis and the acquisition of stress tolerance. Enhanced synthesis of hsps was detected in highly purified T cells and two cloned T cell lines representing helper T (D10) and cytotoxic T cell (Qa-2 128.38) subsets at temperatures as low as 39 degrees C with a maximal response at 41 degrees C. Three major hsps with approximate molecular weights of 110, 90, and 75 were detected in these T cell populations. Western blot analysis using a monoclonal antibody specific for hsp70 indicated that the 75-kDa protein represented hscp70, the cognate or constitutively produced member of the hsp70 family. Although the strongly heat-inducible hsp70 could not be detected in T cells incubated at 41 degrees C by immunoblot analysis, two-dimensional SDS-PAGE analysis did detect a modest induction of hsp70. Thus, hscp70 and not hsp70 was the major intracellular hsp70 member in T cells incubated at febrile temperatures. Enhanced hsp synthesis reflected augmented transcription of hsp genes which was contingent on the continued presence of hyperthermic stress. In order to determine whether induction of hsp synthesis conferred a state of increased resistance to thermal stress, splenic T cells were incubated at either 37 degrees or 41 degrees C (induction temperatures) and then subjected to a heat-shock challenge temperature. These studies revealed that following heat-shock challenge, mitogen-stimulated T cells preincubated at 41 degrees C synthesized DNA at an enhanced rate relative to controls (induction temperature, 37 degrees C). Thus, febrile temperatures were capable of inducing a state of acquired thermotolerance in T cells. However, the thermotolerant state did not protect T cell proliferation against other unrelated stressors such as cadmium and dexamethasone. Reconstitution experiments with accessory cells and interleukin-2-containing supernatants failed to reveal enhanced resistance in thermotolerant T cells to cadmium toxicity or the immunosuppressive activities of dexamethasone. The possibility that higher intracellular concentrations of hsps are required to demonstrate protection against these stressors was tested by the concurrent exposure of T cells to a febrile temperature (41 degrees C) and ethanol. This resulted in a synergistic increase in hsp90 and hsp70 synthesis; however, there was no evidence of enhanced resistance to cadmium- or dexamethasone-induced stress in T cells given this induction protocol. Similarly, alloreactive cytotoxic T lymphocyte responses were inhibited to the same extent in both control and thermotolerant T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses.

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.

Antigen-presenting B cells: efficient uptake and presentation by activated B cells for induction of cytotoxic T lymphocytes against vesicular stomatitis virus.

We have evaluated the efficacy of mitogen (LPS/DxSO4)-activated B cells (B lymphoblasts) to function as antigen-presenting cells (APC) for vesicular stomatitis virus (VSV). Our studies revealed that B lymphoblasts induced potent cytotoxic thymus (T)-derived lymphocyte (CTL) activity in VSV-immune splenic T cells depleted of adherent accessory cells. Dose-response curves indicated that B lymphoblasts were approximately 15-20 times more efficient APC than spleen cells for CTL induction against VSV. There was little evidence of reprocessing of viral antigens by the responder population because only CTL activity restricted to the parental haplotype of the B lymphoblast was generated following stimulation of VSV-immune F1 T cells. B lymphoblasts activated VSV-specific memory CTL which expressed the Lyt-1-23+, AsGM1+ phenotype without activating natural killer and/or lymphokine-activated killer cells. The ability of B lymphoblasts to function as efficient APC was not related to enhanced viral replication in these cells because potent VSV-specific proliferative and class I-restricted CTL responses were induced by B lymphoblasts infected with VSV rendered noninfectious by exposure to ultraviolet (uv) light. This indicates that activated B cells can efficiently process and present input virion protein. Purified splenic B cells that were not activated by mitogen stimulation did not function as APC for VSV even at high multiplicities of infection. The failure of B cells to function as APC for VSV was related to inefficient uptake of VSV and their inability to provide accessory cell signals required for T-cell proliferation; both these functions developed following mitogen stimulation. These data suggest that activated B cells may function as a potent APC population for virus independent of the specificity of their immunoglobulin antigen receptor.

Analysis of T-cell subset proliferation at afebrile and febrile temperatures: differential response of Lyt-1+23- lymphocytes to hyperthermia following mitogen and antigen stimulation and its functional consequence on development of cytotoxic lymphocytes.

Poikilotherms are now known to increase their survival by behaviorally induced fevers in response to pathogenic infection. Increased host resistance to viral and bacterial infections has also been noted in homeotherms whose body temperature has been elevated by manipulation of ambient temperature. These observations suggest that fever may increase host resistance by augmenting acquired immunity; thus, this highly conserved response during evolution may provide a survival advantage against environmental pathogens. This possibility has prompted us to investigate the influence of a temperature characteristic of a modest fever in humans (39 degrees C) on T-cell proliferation and function. Our studies revealed that T-cell mitogenesis was enhanced when cultures were incubated at the febrile temperature (39 degrees C). Analysis of T-cell subsets demonstrated that temperature enhanced the mitogenic (Concanavalin A) response of Lyt-1+23- splenocytes; in contrast, hyperthermia was deleterious to lectin-driven proliferation of the Lyt-1-23+ population even in the presence of large quantities of recombinant interleukin-2 (rIL-2). B-cell mitogenesis was invariably inhibited by hyperthermia over a broad range of concentrations of lipopolysaccharide (LPS). Although T-cell mitogenesis was enhanced at the febrile temperature, T-cell proliferation induced by alloantigens or by a murine pathogen, Sendai virus (SV), was diminished at the febrile temperature. Hyperthermia inhibited SV-induced proliferation of Lyt-1+23- lymphocytes, indicating that a febrile temperature can either augment or inhibit T-cell proliferation of the same T-cell subset depending upon the activation signal (i.e., lectin or antigen). Because effector cell development depends upon antigen-induced clonal expansion (proliferation), we evaluated the influence of temperature on primary cytotoxic thymus (T)-derived lymphocyte (CTL) responses against alloantigens and secondary CTL responses against SV under afebrile and febrile conditions. We consistently observed that the induction of alloreactive and virus-specific CTL was diminished in cultures incubated at the elevated temperature, suggesting that a thermosensitive event(s) exists in the progression of CTL derived from either CTL precursors (CTLp) or memory CTL. Furthermore, hyperthermia reduced the number of SV-specific CTL detectable by limiting dilution analysis, suggesting that another event independent of clonal expansion was thermolabile during effector cell development. In view of these results, we suggest that it may be premature to conclude that the observed increase in host resistance induced by a febrile state is mediated by enhanced cell-me

Growth inhibition of a B cell leukemia: evidence implicating an anti-idiotype immune response for protective tumor immunity.

BCL1, a spontaneous surface IgM (mu lambda)-positive (sIgM+) B cell leukemia of BALB/c (Igha) origin rarely grows in the Ig heavy chain (Igh) congenic mouse C.B-20 (Ighb) but is highly metastatic and lethal in the host strain of origin. Previous studies indicated that BCL1 tumor immunity in C.B-20 mice was associated with a T cell-mediated immune response against H-40, a minor histocompatibility (H) antigen controlled by a gene linked to the Igh locus. However, we observed that BCL1 leukemia grew progressively in BAB-14 (Igha/b) mice, a strain capable of generating an anti-H-40 immune response. This suggested that anti-H-40 immunity was insufficient for protection and implied that an Igh-V (variable) region gene product was also important for BCL1 growth inhibition. We therefore evaluated the role of two possible Igh-V region-linked gene products in BCL1 growth inhibition; namely, an Igh-V region-linked minor H antigen or alternatively the BCL1 IgM idiotype (Id). We could find no evidence for an Igh-V region-linked minor H antigen because immunosuppressed (500 R) CB-20 mice reconstituted with C.B-20 anti-BAB-14 splenocytes were susceptible to BCL1 growth, whereas recipients reconstituted with C.B-20 anti-BALB/c splenocytes were resistant to BCL1 challenge. In contrast, C.B-20 mice immunized against purified BCL1 IgM protein could adoptively confer BCL1 tumor immunity. C.B-20 mice immunized against other BALB/c IgM myeloma proteins containing either lambda or kappa light chains failed to protect C.B-20 mice suggesting that recognition of a unique determinant (Id) and not an allotype was crucial for tumor immunity. The BCL1 mu-chain appeared to make the major contribution to the idiotypic determinant because a hybridoma product composed of BCL1 mu-chains and BALB/c kappa-chains still elicited BCL1 immunity. Adoptive transfer of C.B-20 anti-BCL1 Id splenocytes into irradiated recipients that prevented an anti-H-40 response due to H-40 tissue expression failed to adoptively confer BCL1 immunity. Thus, these data suggest that BCL1 growth inhibition requires a T cell-mediated response against both H-40 and the BCL1 Id; these responses must be elicited concurrently in the tumor-bearing host to achieve protective BCL1 immunity.

Immune rejection mechanisms in murine leukemia. I. Timing of tumor cell rejection process relative to the development of humoral and cell-mediated cytotoxic immune responses.

Studies were undertaken to investigate the relationship between cell-mediated and humoral immune responses in the rejection of L1210/MTX-Rev (LR) leukemia cells in CD2F1 mice. The anti-LR antibody (humoral) response was defined by both its cytotoxic antibody titer and isotype composition, assessed by a complement-dependent cytotoxicity assay and radioimmune assay, respectively. The cytotoxic thymus (T)-derived lymphocyte response in the spleen was quantitated by 125I release by 125I-IUdR-labelled target cells. Analysis of the sera of tumor-bearing mice indicated that the LR leukemia cells elicited a wide spectrum of anti-tumor antibody isotypes. In mice that mounted a rejection response, the IgM anti-LR response was transient, while in those that did not, the IgM response persisted. Antibody titers for all isotypes remained low until the onset of LR rejection. At that time, high titers of IgG anti-LR antibodies, predominantly of the IgG2a subclass, were detected in sera of tumor-free mice. Thus, the LR rejection response coincided best with the appearance of high titers of IgG2a anti-LR antibodies. A single i.p. injection of viable LR cells elicited a potent cell-mediated immune response; neither the appearance nor the magnitude of the cell-mediated immune response as measured in the spleen correlated well with the onset or the strength of the LR rejection response in the peritoneal cavity. The LR peritoneal cell population grew unabated in the presence of an intense spleen cell-mediated immune response, and the rejection process began at a time when little cellular immunity could be detected. These results suggest that in the rejection response IgM antibodies contribute little, if any, to the process, and the role of cytotoxic T lymphocytes is questionable (uncertain). The rejection of LR cells appears to be primarily mediated by IgG2a antibodies.

Cadmium-induced suppression of the primary immune response and acute toxicity in mice: differential interaction of zinc.

Four groups of 6-week-old C3H mice were injected sc with either sterile saline, 2.8 mg Cd/kg body weight, 2.8 mg Zn/kg body weight, or 2.8 mg each of Cd and Zn/kg body weight. Forty-eight hours after the initial injection, all mice received a second dose of their respective treatments plus an iv injection of sheep red blood cells. On Days 2, 3, 4, and 5 postimmunization the mice were killed. Spleen cells were used in a hemolytic plaque-forming assay for the quantitation of the primary humoral response. Although the combined administration of zinc and cadmium completely prevented the fatal effects of the cadmium (0 vs 55% mortality), zinc failed to alleviate the cadmium-induced inhibition of the humoral response.