A critical role for CD8 T cells in a nonhuman primate model of tuberculosis

Abstract

The role of CD8 T cells in anti-tuberculosis immunity in humans remains unknown, and studies of CD8 T cell-mediated protection against tuberculosis in mice have yielded controversial results. Unlike mice, humans and nonhuman primates share a number of important features of the immune system that relate directly to the specificity and functions of CD8 T cells, such as the expression of group 1 CD1 proteins that are capable of presenting Mycobacterium tuberculosis lipids antigens and the cytotoxic/bactericidal protein granulysin. Employing a more relevant nonhuman primate model of human tuberculosis, we examined the contribution of BCG- or M. tuberculosis-elicited CD8 T cells to vaccine-induced immunity against tuberculosis. CD8 depletion compromised BCG vaccine-induced immune control of M. tuberculosis replication in the vaccinated rhesus macaques. Depletion of CD8 T cells in BCG-vaccinated rhesus macaques led to a significant decrease in the vaccine-induced immunity against tuberculosis. Consistently, depletion of CD8 T cells in rhesus macaques that had been previously infected with M. tuberculosis and cured by antibiotic therapy also resulted in a loss of anti-tuberculosis immunity upon M. tuberculosis re-infection. The current study demonstrates a major role for CD8 T cells in anti-tuberculosis immunity, and supports the view that CD8 T cells should be included in strategies for development of new tuberculosis vaccines and immunotherapeutics.

Figure 1. CD8 Ab treatment of BCG-vaccinated macaques resulted in profound depletion of CD8 lymphocytes and mycobacterium-specific CD8 T effector cells during M. tuberculosis infection.

(A) Flow cytometry data showed that anti-CD8 Ab-treated BCG-vaccinated macaques, but not isotype control IgG-treated BCG-vaccinated or naïve saline-treated macaques, exhibited marked decreases in percentages and absolute (data not shown) numbers of CD3+CD8+ and CD3−CD8+ lymphocytes in blood (upper left) and BAL fluid (upper right) during M. tuberculosis infection. Means for six animals are shown for each group, and error bars represent SEM. Arrows indicate the times for the treatment with anti-CD8 Ab or control antibodies. (B) Intracellular cytokine staining data showed that anti-CD8 Ab-treated BCG-vaccinated macaques exhibited an absence of PPD-specific IFNγ-producing CD8 T cells early after M. tuberculosis infection.

Figure 2. Depletion of CD8 lymphocytes resulted in a significant decrease in BCG-induced immune control of M. tuberculosis.

(A) CD8 Ab-treated macaques exhibited higher levels of bacilli in BAL fluid than isotype IgG-treated macaques after pulmonary M. tuberculosis infection. Data are means +/− SEM of CFU counts/10 ml BAL fluid derived from 6 macaques for each group; * indicates p<0.05 for a comparison between the CD8 Ab-treated and isotype IgG-treated group. (B) CD8 Ab-treated macaques showed significantly higher levels of bacilli organisms in lung tissues than isotype control macaques after pulmonary M. tuberculosis infection. Data are mean values with SEM error bars of CFU counts/1 ml lung tissue homogenates derived from 6 macaques for each group. Rt, right; Lt, left. Note that right caudal lobe was the M. tuberculosis infection site for each macaque; *, p<0.05 (by both ANOVA and nonparametric t test) for a comparison between the CD8 Ab-treated and isotype control IgG-treated groups.

Figure 3. Depletion of CD8 lymphocytes in CD8 Ab-treated macaques resulted in a significant decrease in BCG vaccine-induced immunity against tuberculosis after M. tuberculosis infection.

(A) Top-panel photos show that the isotype IgG-treated BCG-vaccinated macaque (7406) exhibited limited numbers of granulomas (small arrows) in the cut-section of the right caudal lobe; the CD8 Ab-treated macaque (7348) showed extensive tuberculosis granulomas (large arrows) in the right caudal lobe, with a unilaterally enlarged hilar lymph node (a large green arrow). A naïve macaque (7419) showed large extensive tuberculosis lesions with caseation (large arrows), and much larger bilateral hilar lymph nodes (large green arrows). Middle- and bottom-panel photos show tuberculosis lesions in cut-sections of lungs of six monkeys in three respective groups. Extent and severity of the lesions could be adjudged based on the examples pointed by arrows at the top panel photos. Overall, CD8 Ab-treated macaques exhibited greater numbers of lung lobes displaying extensive coalescing granulomas than isotype IgG-treated control animals (p<0.05, by nonparametric t test). The CD8 Ab-treated macaques also showed more lobes with extensive caseating and miliary lesions or caseation pneumonia than the isotype IgG-treated controls (p<0.05, by nonparametric t test). Tuberculosis lesions in the CD8 Ab-treated macaques were more likely distributed or disseminated in other lobes or opposite lungs and hilar nodes/pleural. Small vertical/horizontal bars at bottom-left corner of each photo represent the 1-cm scale derived from the fluorescence rulers of each original photo. (B) Gross pathology scores of tuberculosis lesions in CD8 Ab-treated group, Isotype control IgG-treated group and naïve saline-treated group of macaques. Each macaque was given a total gross pathology scores based on the tuberculosis lesions in thoracic and extrathoracic organs. The mean gross pathology score was then calculated for each group of macaques and subjected to statistical analysis. *, P<0.05 (by ANOVA and nonparametric t test) for comparison between CD8 Ab-treated and isotype control IgG-treated groups, and for comparison between CD8 Ab-treated and naïve groups. (C) Histology evaluation of lung tissue sections of CD8 Ab-treated, isotype control IgG-treated and naïve saline-treated macaques. Shown are H&E stained sections taken from four representative macaques for each group, with macaque ID and magnification indicated for each image. Note that the isotype control IgG-treated macaques exhibited well-contained granulomas, which were generally infiltrated by numerous lymphocytes and some neutrophils. The CD8-depleted macaques displayed less-contained granulomas that were more likely to be necrotic (arrows). Naïve macaques showed less lymphocytic and more necrotic tuberculosis lesions than the other two groups.

Figure 4. Depletion of CD8 lymphocytes in macaques immunized by previous M. tuberculosis infection resulted in a loss of immune control of tuberculosis following re-infection.

(A) Anti-CD8 Ab treatment of the M. tuberculosis-immunized macaques resulted in profound depletion of CD8 lymphocytes in M. tuberculosis re-infection. Shown are absolute levels of blood CD8 cells (/ul) and BAL fluid CD8 cells (×50,000). (B) Depletion of CD8 lymphocytes in the M. tuberculosis-immunized macaques led to higher levels of bacilli in BAL fluids and increased M. tuberculosis RNA in tissues following M. tuberculosis re-infection by aerosol. Numbers of bacilli are shown as CFU counts in 10 ml of BAL fluid; M. tuberculosis RNA was determined as Ag85B RNA copy numbers in 10 mg of tissue ,,. (C) The CD8 Ab-treated macaques with CD8 depletion developed severe forms of tuberculosis after M. tuberculosis re-infection by aerosol. Macaque ID are indicated for CD8 Ab-treated (right) and isotype control IgG-treated (left) macaques. Shown are the lung surface images of two representative macaques (partially cut in the lung of macaque 2762). Note that the CD8 Ab-treated macaques exhibited pale-colored lungs with dissemination of >0.5 cm coalescing or caseating granulomas or tubercle nodules (the lesion-containing areas are pointed out by large arrows on surface view from 3050) and apparent caseation necrosis. The control macaques displayed no or few small non-caseating granulomas as indicated by small arrows in the lungs. When >0.5 cm coalescing or caseating tubercle nodules both lungs were scored using the scoring system as described in Methods and for the individual macaques, the pathology scores (mean±SD = 72±24) of the CD8-depleted macaques were significantly worse than those (mean±SD = 6±2) of the control animals (p<0.01, by nonparametric t test). (D) Histology of representative granulomas seen in CD8 Ab-treated (right panel) and isotype control IgG-treated (left panel) macaques. Shown are H&E stained lung sections, with macaque ID and magnification indicated in each slide. Note that granulomas in isotype IgG-treated macaques were small, and highly lymphocytic without apparent necrosis. Granulomas in CD8 Ab-treated macaques were large and less lymphocytic, with necrosis seen in the center as pointed out by arrows.