Plasmodium chabaudi adami: use of the B-cell-deficient mouse to define possible mechanisms modulating parasitemia of chronic malaria.

Our previous observation that B-cell-deficient JH-/- mice utilize T cell-dependent immunity to suppress acute Plasmodium chabaudi adami-induced malaria but then develop chronic low-level parasitemia prompted this study of control mechanisms for chronic parasitemia. When we infected JH-/- mice with blood-stage parasites, chronic parasitemia exacerbated after the 6th month and persisted for up to 17 months. This exacerbation of parasitemia could not be attributed to host aging because the time-course of acute infection in naïve aged mice was nearly identical to that seen in young mice. Nor could exacerbated parasitemia be attributed to mutation in the parasite genome resulting in increased virulence; when subinoculated into naïve JH-/- mice, parasites from chronically infected JH-/- mice with exacerbated parasitemia produced acute stage parasitemia profiles in most recipients comparable to those seen in JH-/- mice upon infection with the original stabilate material. Of the pro-inflammatory cytokines measured, including IFNgamma, TNFalpha, IL-12p70, and MCP-1beta, none were significantly different in the sera of mice with exacerbated parasitemia compared to uninfected controls. Levels of IL-6 were significantly (P=0.002) less in the sera of mice with exacerbated parasitemia. Serum levels of the anti-inflammatory cytokine, TGFbeta, were significantly depressed in chronically infected JH-/- mice compared to uninfected controls. In contrast, IL-10 levels were markedly increased. These findings suggest that the cytokine balance may be disturbed during chronic malaria, thereby impacting on mechanisms that modulate levels of parasitemia.

DNA vaccines against chronic lung infections by Pseudomonas aeruginosa.

Vaccines containing outer membrane protein F (OprF) of Pseudomonas aeruginosa are effective in reducing lesion severity in a mouse pulmonary chronic infection model. One OprF-based vaccine, called F/I, contains carboxy oprF sequences fused to oprI in an expression vector. When delivered three times biolistically by gene gun, the F/I vaccine induces protection that is antibody-mediated in outbred mice. To demonstrate the role of F/I-induced antibody-mediated immunity, B-cell-deficient [B(-)] and B-cell-intact [B(+)] mice were immunized with F/I, challenged with Pseudomonas, and examined for lesion severity. As expected, F/I-immunized B(+) mice had fewer and less severe lesions than vector-immunized B(+) mice. However, surprisingly, F/I- and vector-immunized B(-) mice were equally protected to levels similar to F/I-immunized B(+) mice. Examination of immune cell populations and cytokine levels indicated a relative increase in the quantity of CD3+ T-lymphocytes in vector- or F/I-immunized and challenged B(-) mice compared to B(+) mice. These data indicate the protective role played by cell-mediated immunity in B(-) mice, which supports our hypothesis that cell-mediated immunity can play an important role in protection against P. aeruginosa.

CD8(+)-T-cell depletion ameliorates circulatory shock in Plasmodium berghei-infected mice.

The Plasmodium berghei-infected mouse model is a well-recognized model for human cerebral malaria. Mice infected with P. berghei exhibit (i) metabolic acidosis (pH < 7.3) associated with elevated plasma lactate concentrations, (ii) significant (P < 0.05) vascular leakage in their lungs, hearts, kidneys, and brains, (ii) significantly (P < 0.05) higher cell and serum glutamate concentrations, and (iv) significantly (P < 0.05) lower mean arterial blood pressures. Because these complications are similar to those of septic shock, the simplest interpretation of these findings is that the mice develop shock brought on by the P. berghei infection. To determine whether the immune system and specifically CD8(+) T cells mediate the key features of shock during P. berghei malaria, we depleted CD8(+) T cells by monoclonal antibody (mAb) treatment and assessed the complications of malarial shock. P. berghei-infected mice depleted of CD8(+) T cells by mAb treatment had significantly reduced vascular leakage in their hearts, brains, lungs, and kidneys compared with infected controls treated with rat immunoglobulin G. CD8-depleted mice were significantly (P < 0.05) protected from lactic acidosis, glutamate buildup, and diminished HCO(3)(-) levels. Although the blood pressure decreased in anti-CD8 mAb-treated mice infected with P. berghei, the cardiac output, as assessed by echocardiography, was similar to that of uninfected control mice. Collectively, our results indicate that (i) pathogenesis similar to septic shock occurs during experimental P. berghei malaria, (ii) respiratory distress with lactic acidosis occurs during P. berghei malaria, and (iii) most components of circulatory shock are ameliorated by depletion of CD8(+) T cells.

Assessing vascular permeability during experimental cerebral malaria by a radiolabeled monoclonal antibody technique.

Vascular endothelial integrity, assessed by Evans blue dye extrusion and radiolabeled monoclonal antibody leakage, was markedly compromised in the brain, lung, kidney, and heart during Plasmodium berghei infection, a well-recognized model for human cerebral malaria. The results for vascular permeability from both methods were significantly (P < 0.001) related.

Nitric oxide is neither necessary nor sufficient for resolution of Plasmodium chabaudi malaria in mice.

Malaria is a life-threatening re-emerging disease, yet it is still not clear how bloodstage malarial parasites are killed. Nitric oxide (NO), which has potent anti-microbial activity, may represent an important killing mechanism. The production of NO during descending Plasmodium chabaudi parasitemia, a period when parasites are killed by the immune response, supports this concept. However, NOS20/0 and NOS30/0 mice as well as mice treated with NO synthase 2 (NOS2) inhibitors do not develop exacerbated malaria, indicating that NO production is not necessary for the suppression of P. chabaudi parasitemia. It is possible due to the plasticity in the immune response during malaria that Ab-mediated immunity is enhanced in the absence of NO, thereby explaining the lack of exacerbated malaria in NOS-deficient mice even though NO may function in protection. However, NOS2- and B cell-deficient mice, which cannot use Ab-mediated immunity, suppress their parasitemia with a similar time course as B cell-deficient controls. C57BL/6 mice treated with Propionibacterium acnes to elicit high levels of macrophage-derived NO have a similar time course of P. chabaudi parasitemia as P. acnes-treated NOS20/0 mice, which do not produce NO; this indicates that NO is not sufficient for parasite killing. Collectively, these results indicate that NO is not necessary or sufficient to resolve P. chabaudi malaria.

Role of inducible nitric oxide synthase in the regulation of VCAM-1 expression in gut inflammation.

The objectives of this study were to assess the role of the inducible isoform of nitric oxide synthase (iNOS) on vascular cell adhesion molecule 1 (VCAM-1) expression in vivo in an acute model of inflammation induced in iNOS-deficient (iNOS-/-) mice and compare these data to those obtained by pharmacological inhibition of iNOS in a CD4+ T lymphocyte-dependent model of chronic colitis. VCAM-1 expression was quantified in vivo using the dual radiolabel monoclonal antibody technique. We found that intraperitoneal injection of 10 microg/kg tumor necrosis factor-alpha (TNF-alpha) enhanced VCAM-1 expression by approximately twofold in the colon, cecum, and stomach but not small intestine in iNOS-/- mice compared with TNF-alpha-injected wild-type mice. Injection of wild-type mice with 25 microg/kg TNF-alpha further enhanced VCAM-1 expression by approximately twofold compared with wild-type mice injected with 10 microg/kg TNF-alpha; however, VCAM-1 expression was not further enhanced in any gastrointestinal organ system in iNOS-/- mice. In a second series of experiments, we found that continuous inhibition of iNOS using oral administration of NG-iminoethyl-L-lysine did not alter the enhanced levels of VCAM-1 expression in the colon nor did it alter the severity of colonic inflammation in SCID mice reconstituted with CD4+, CD45RB(high) T cells. We conclude that iNOS may regulate VCAM-1 expression in acute inflammation; however, this effect is modest and tissue specific and occurs only when VCAM-1 expression is submaximal. iNOS does not appear to modulate VCAM-1 expression in an immune model of chronic colitis.

Gamma delta T-cell function in pathogenesis of cerebral malaria in mice infected with Plasmodium berghei ANKA.

Mice depleted of gammadelta T cells by monoclonal antibody treatment and infected with Plasmodium berghei ANKA did not develop cerebral malaria (CM). In striking contrast, delta0/0 mice infected with P. berghei developed CM despite their gammadelta T-cell deficiency. gammadelta T cells appear to be essential for the pathogenesis of CM in mice having experienced normal ontogeny but not in mice genetically deprived of gammadelta T cells from the beginning of life.

Flow cytometric quantitation of yeast a novel technique for use in animal model work and in vitro immunologic assays.

Animal models of fungal and other infectious diseases often require that the number of organisms in tissue be quantified, traditionally by grinding organs, plating them on agar and counting colony forming units (CFU). This method is labor intensive, slow as some fungi require two weeks of culture and limited in reliability by poor plating efficiency. To circumvent these problems, we developed a flow cytometric method to quantify yeast. In vitro cultured Blastomyces dermatitidis, Cryptococcus neoformans, Candida albicans and Histoplasma capsulatum yeast were labelled with specific monoclonal or polyclonal antibodies to stain surface determinants or with Calcofluor to stain cell-wall chitin. A defined number of fluorescently labelled beads were added prior to acquisition by flow cytometry as a reference standard for quantitation. Beads were readily distinguished from yeast by forward scatter, side scatter and intensity of fluorescence. Cultured yeast were enumerated by both standard CFU determination and flow cytometry in a range of 10(2) to 10(7) cells. Only flow cytometry enabled discrimination of live and dead yeast by using appropriate fluorescent dyes. The flow cytometric method was applied to murine models of histoplasmosis and blastomycosis to quantify the burden of fungi in the lungs of infected mice. Labelling yeast with Calcofluor alone resulted in unacceptably high levels of nonspecific binding to mouse cell debris. In contrast, labelling H. capsulatum with a rabbit polyclonal antiserum and B. dermatitidis with a monoclonal antibody to the surface protein WI-1 permitted accurate quantitation. We conclude that this flow cytometry technique is rapid, efficient and reliable for quantifying the burden of infection in animal models of fungal disease. The technique also should lend itself to performing cytotoxicity assays that require discrimination of live and dead fungi, or phagocytosis assays that require discrimination of intracellular and extracellular organisms.

The time course of selected malarial infections in cytokine-deficient mice.

Murine malarial parasites have long been characterized by their requirement for either antibody-mediated immunity (AMI) or cell-mediated immunity (CMI) for suppression of acute parasitemia, with Plasmodium yoelii reportedly requiring AMI for suppression and P. chabaudi requiring CMI. To assess this characterization in terms of the current T(H1)/T(H2)-CMI/AMI hypothesis, we infected gene-targeted "knockout" mice lacking either a type-1 cytokine (IL-2 or IFN-gamma) or a type-2 cytokine (IL-4 or IL-10) with one or the other species of Plasmodium. We observed that type-1 cytokine-deficient mice developed exacerbated malaria with either P. yoelii or P. chabaudi, compared with that seen in heterozygote controls. Moreover, type-2 cytokine knockout mice showed a similar time course of infection with either parasite compared with that seen with their controls. We conclude that the mechanism of resolution of these well characterized malarial infections cannot be linked definitely to these T(H1)- and T(H2)-associated cytokines as predicted by the T(H1)/T(H2)-CMI/AMI hypothesis.

Human gamma delta T cell subset-proliferative response to malarial antigen in vitro depends on CD4+ T cells or cytokines that signal through components of the IL-2R.

We examined the cellular and molecular basis of the proliferative response of human gamma delta T cells in cultures of PBMC stimulated with blood-stage Plasmodium falciparum malarial Ag. Flow cytometry revealed that maximal gamma delta T cell proliferation occurs after maximal CD4+ alpha beta T cell proliferation. Depletion of CD4+ T cells from PBMC before stimulation with malarial Ag markedly reduces the number of proliferating gamma delta T cells, which suggests that CD4+ T cells function in providing help to gamma delta T cells to respond to this parasite Ag. Removal of gamma delta T cells, however, did not alter the expansion of the CD4+ T cell subset. The addition of exogenous IL-2, IL-4, or IL-15 restored the capacity of gamma delta T cells to proliferate in Ag-stimulated cultures of PBMC depleted of CD4+ T cells. mAbs specific for the alpha- and beta-subunits of the IL-2 receptor inhibit the gamma delta T cell subset expansion in cultures stimulated with malarial Ag. Taken together, these findings suggest that the proliferation of gamma delta T cells in response to malarial Ag is dependent on the presence of CD4+ alpha beta T cells, but the requirement for CD4+ alpha beta T cells can be met by cytokines that use the IL-2R.

Expansion of the gammadelta T cell subset in vivo during bloodstage malaria in B cell-deficient mice.

Mice rendered B cell-deficient either by chronic anti-mu treatment initiated at birth or by gene knockout (JHD and mu-MT mice) suppressed acute Plasmodium chabaudi infections with a time course similar to intact control mice. Moreover, both kinds of B cell-deficient mice showed a 50- to 100-fold increase in splenic gammadelta T cell number after suppression of parasitemia compared with uninfected B cell-deficient controls; the magnitude of this increase resulted in significantly (P< 0.05) greater numbers of splenic gammadelta T cells in the B cell-deficient mice than in infected B cell-intact controls (about 10-fold). In contrast, the number of splenic CD4+ alphabeta T cells was only slightly elevated (< 2-fold) in both kinds of B cell-deficient mice compared with their intact controls. The number of splenic gammadelta T cells following suppression of P. vinckei parasitemia was approximately ninefold greater in JHD mice than in C57BL/6 controls, whereas similar numbers of splenic CD4+ alphabeta T cells were detected. Maximal numbers of gammadelta T cells were in cell-cycle in both JHD and C57BL/6 mice during descending P. chabaudi parasitemia, but the number of gammadelta T cells in cell-cycle was greater in B cell-deficient mice than in intact controls. Interleukin-10 (IL-10), a potent TH1 cell-suppressive molecule, does not appear to down-regulate the gammadelta T cell response during malaria in B cell-intact mice because the magnitude of the gammadelta T cell response was not significantly greater in IL-10 knockout mice compared with heterozygote controls. These findings collectively indicate that a markedly enhanced expansion of the gamma delta T cell population occurs in the absence of B cells, and this expansion occurs predominantly during acute malaria when parasite burdens are similar in B cell-deficient animals and intact controls.

Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria.

We determined the requirement for selected lymphocyte subsets and cytokines in the pathogenesis of experimental murine cerebral malaria (CM) by using gene-targeted knockout and mAb-suppressed mice. Plasmodium berghei ANKA infection induced CM in A 0/0 mice, which lack expression of surface MHC class II glycoproteins and consequently express a severe and chronic reduction in numbers of CD4+ T cells. However, when A 0/0 mice, which are on a C57BL/6 x 129 genetic background, or immune-intact C57BL/6 controls treated with anti-CD4 mAb were infected, none developed CM. The latter finding confirms an earlier report that CD4+ T cells are required for CM to occur and additionally indicates that the reduced numbers of CD4+ T cells present in A 0/0 mice are sufficient for CM development. Neither the recently described CD4+, NK1.1+ T cell subset shown to be present in A 0/0 mice nor traditional NK cells seem to be required for the induction of CM because A 0/0 and C57BL/6 mice severely depleted of both NK1.1+ populations with mAb developed CM as readily as did normal Ig-treated controls. Deficiency of Th1-associated cytokines (IFN-gamma or IL-2) in mice by gene-targeted disruptions completely inhibited CM development, whereas the lack of Th2-associated cytokines (IL-4 or IL-10) did not prevent this disease. Our observation that B cell-deficient JHD and microMT mice developed CM provides evidence that neither B cells, their products, nor B cell Ag presentation are a requisite for CM pathology. We further observed that neither beta 2m 0/0 knockout mice, which lack CD8+ alpha beta T cells, nor C57BL/6 mice depleted of CD8+ T cells with anti-CD8 mAb treatment developed CM, leading us to conclude that CD8+ T cells are also crucial for the development of CM.

Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum.

Flow cytometry was evaluated as a method of assessing in vitro the effects of leukocytes on blood-stage Plasmodium falciparum. Hydroethidine is converted by metabolizing cells to ethidium, a nucleic acid fluorochrome. After incubation with hydroethidine, viable and dead leukocytes and parasitized and uninfected erthrocytes could all be identified on the basis of fluorescence intensity and size. Leukocytes can therefore be eliminated from further analysis; this allows assessment, at any parasite developmental stage, of the level of parasitemia within erythrocytes in the presence of any of several types of leukocytes. Whether leukocytes actually kill intraerythrocytic parasites can therefore be determined and the level of cytotoxicity can be assessed. The ability of leukocytes to prevent merozoites from invading new erythrocytes, i.e., inhibition of parasite invasion, can also be assessed by this method. When erythrocytes containing schizont-stage parasites were cocultured with different leukocyte populations and the level of parasitemia was determined after merozoite release and invasion, only cultures containing gamma delta T cells inhibited parasite invasion. The different blood-stage forms of the parasite vary in nucleic acid content, which allows each of the developmental stages to be distinguished by flow cytometry; this permits assessment of changes in parasite development in the presence of leukocytes. Monocyte-derived macrophages (MDMs) appeared to have an effect on parasite development. In this instance, when erythrocytes containing ring-form parasites were cocultured with MDMs and harvested 24 h later, the parasites in cultures containing MDMs were at the late schizont stage, whereas parasites in control cultures were early trophozoites; this finding suggests that MDMs accelerate parasite development. Together, these results indicate that flow cytometry is potentially useful for measuring the following effects mediated by leukocytes: (i) level of cytotoxicity, (ii) changes in parasite development, and (iii) inhibition of parasite invasion.

Gamma delta T cell-induced nitric oxide production enhances resistance to mucosal candidiasis.

Despite the prevalence of gamma delta T cells in mucosae that are typically colonized by Candida albicans, little is known of the possible role of these cells in resistance to candidiasis. A sharp increase in the number of gamma delta T cells and macrophages following intraperitoneal inoculation of mice with C. albicans led us to examine the role of these cells in the immune response to C. albicans. We show that the gamma delta T cells enhance macrophage nitric oxide (NO) production and anti-candida activity, in vitro. We also propose that the gamma delta T cells regulate macrophage function during candidiasis in vivo as well, because depletion of these cells abrogated inducible NO synthase expression in mucosae and enhanced murine susceptibility to candidiasis.

Gamma delta T cells function in cell-mediated immunity to acute blood-stage Plasmodium chabaudi adami malaria.

To determine whether gamma delta T cells are essential for the resolution of acute Plasmodium chabaudi adami (P. c. adami) malaria, we depleted gamma delta T cells from C57BL/6 mice with hamster monoclonal anti-TCR gamma delta Ab treatment. During the period in which control mice that had received normal hamster IgG completely resolved infections, gamma delta T cell-depleted mice were unable to suppress their infections. Because the number of splenic CD4+ alpha beta T cells in these anti-TCR-gamma delta-treated mice with nonresolving malaria was similar to control mice, it appears that CD4+ alpha beta T cells alone cannot mediate early resolution even though they are known to play a critical role in immunity to blood-stage malaria. Mice treated with anti-CD4 mAb also failed to resolve P. c. adami malaria. Depletion of CD4+ alpha beta T cells from the spleens of infected mice resulted in minimal expansion of the splenic CD4- gamma delta T cell subset compared with infected control mice. Together, these findings indicate that activation of the gamma delta T cell subset, which requires the presence of CD4+ alpha beta T cells, is essential for resolution of acute P. c. adami malaria. To determine whether gamma delta T cells require either Abs or B cells to achieve their protective activity, B cell-deficient JHD mice were treated with the same depleting anti-TCR-gamma delta Abs. Whereas control JHD mice injected with hamster IgG resolved acute P. c. adami malaria, JHD mice depleted of gamma delta T cells failed to do so. We conclude that gamma delta T cells suppress P. c. adami parasitemia by mechanisms of immunity independent of Ab and B cells.

Inhibition of Plasmodium falciparum in vitro by human gamma delta T cells.

As assessed by flow cytometry, human gamma delta T cells were shown here to inhibit replication of blood-stage Plasmodium falciparum in vitro in a dose-dependent fashion; no other leukocyte population tested was suppressive. Replication of intraerythrocytic stages of the parasite (rings, trophozoites, and schizonts) was not affected by coculture with gamma delta T cells nor were erythrocytes damaged by this coculture, indicating that the targets recognized by gamma delta T cells are extracellular merozoites in transit to new host erythrocytes. Moreover, parasite inhibition requires contact between gamma delta T cells and merozoites. These findings suggest that gamma delta T cells may exert a protective effect in immunity to malaria.

The resolution of acute malaria in a definitive model of B cell deficiency, the JHD mouse.

Because the role of cell-mediated immunity (CMI) in the resolution of blood-stage malaria remains unclear, we examined the question of whether mice completely lacking Ab-mediated immunity (AMI) but possessing some CMI can resolve experimental malaria previously reported not to require AMI for resolution. Severe combined immunodeficient mice reconstituted with enriched immune T cells (< 0.5% B220+ cells) suppressed acute Plasmodium chabaudi adami parasitemia, suggesting that T, but not B, cells are required to clear this form of malaria. In addition, JHD mice, which are a definitive model of B cell deficiency, were also shown to resolve P. chabaudi adami, Plasmodium vinckei petteri and Plasmodium chabaudi chabaudi malaria. These observations collectively establish that CMI alone can mediate the clearance of acute malaria caused by these subspecies of Plasmodium. Moreover, the protective cell-mediated immune response involved depends upon CD4+ T cells because JHD mice treated with anti-CD4 mAb do not resolve their infections. These results suggest that evaluation of immunization regimens to activate CD4+ T cell dependent cell mediated immunity against Plasmodium falciparum may be appropriate.

Role of CD4+ T cells in the expansion of the CD4-, CD8- gamma delta T cell subset in the spleens of mice during blood-stage malaria.

The previously observed expansion of the splenic gamma delta T cell subset was examined during the course of murine malaria to determine whether CD4+ T cells are required. Flow cytometric analysis during the course of Plasmodium chabaudi adami malaria in both C57BL/6 and BALB/c mice revealed that the maximal percentage of CD4+ T cells that were blasts occurred during the period of ascending parasitemia, whereas the maximal numbers of gamma delta T cells and blast cells occurred during the period of descending parasitemia. Transfer of enriched populations of CD4+ cells (> 75%) containing < 0.9% gamma delta T cells from immune BALB/c donor to SCID mice led to a population of gamma delta T cells that constituted 37% of the splenic T cells in the recipients and allowed them to resolve their infections. Transfer of the CD4- fraction did not suppress parasitemia. These results suggest that CD4+ T cells are activated early during the infection and are required for the subsequent expansion of the gamma delta T cell population. Furthermore, the maximal gamma delta T cell blast response during the period of descending parasitemia and the detection of high levels of these cells only in models that resolved their infections suggest that these cells may function in the resolution of blood-stage malaria.

Resolution of blood-stage malarial infections in CD8+ cell-deficient beta 2-m0/0 mice.

We utilized a definitive model of CD8+ T cell deficiency, the beta 2-microglobulin-deficient (beta 2-m0/0) mouse, to determine whether CD8+ T cells are required in the resolution of blood-stage malaria. In a parallel experiment, C57Bl/6 mice treated with anti-CD8 mAb showed significantly higher levels of parasitemia than untreated C57Bl/6 control mice at several points during the infection. This finding suggests some role for CD8+ cells in containing malaria. However, the beta 2-m0/0 mice, which are genetically blocked from expressing MHC class I or class Ib glycoproteins and therefore have < 2.5% of the normal number of CD8+ T cells, nevertheless resolved infections with three virulence variants of murine Plasmodium. The resolution of Plasmodium chabaudi adami, Plasmodium yoelii yoelii 17X, and Plasmodium chabaudi chabaudi AS infections by beta 2-m0/0 mice in the virtual absence of CD8+ cells demonstrates that these cells are not required to suppress murine malaria and that the suppression mechanism is not MHC class I restricted. The similarity of the time-course for resolution of infection in beta 2-m0/0 and intact control mice with all three subspecies of Plasmodium further supports the lack of a requirement for CD8+ T cells in the suppression of malarial parasitemia.

Expansion of the CD4-, CD8- gamma delta T cell subset in the spleens of mice during non-lethal blood-stage malaria.

Splenic gamma delta T cells (CD4-, CD8-) increased more than 10-fold upon resolution of either Plasmodium chabaudi adami or P.c. chabaudi infections in C57BL/6 mice compared to controls. Similarly, a 10- to 20-fold expansion of the gamma delta T cell population was observed in beta 2-microglobulin deficient (beta 2-m0/0) mice that had resolved P.c. adami, P.c. chabaudi or P. yoelii yoelii infections. In contrast, increases in the number of splenic alpha beta T cells in these infected mice were only two to three-fold indicating a differential expansion of the gamma delta T cell subset during malaria. Because nucleated cells of beta 2-m0/0 mice lack surface expression of major histocompatibility complex class I and class Ib glycoproteins, our findings suggest that antigen presentation by these glycoproteins is not necessary for the increasing number of gamma delta T cells. Our observation that after resolution of P.c. adami malaria, C57BL/6 mice depleted of CD8+ cells by monoclonal antibody treatment had lower numbers of gamma delta T cells than untreated controls suggests that the demonstrated lack of CD8+ cells in beta 2-m0/0 mice does not contribute to the expansion of the gamma delta T cell population during non-lethal malaria.